Dna detection methods for site specific nuclease activity

ABSTRACT

The present disclosure provides methods for detecting and identifying plant events that contain precision targeted genomic loci, and plants and plant cells comprising such targeted genomic loci. The method can be deployed as a high throughput process utilized for screening the intactness or disruption of a targeted genomic loci and optionally for detecting a donor DNA polynucleotide insertion at the targeted genomic loci. The methods are readily applicable for the identification of plant events produced via a targeting method which results from the use of a site specific nuclease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 61/736,856, filed Dec. 13, 2012. The contents of the entirety ofeach of the foregoing are hereby incorporated in their entireties hereinby this reference.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII formatted sequence listing with a file named“DNA DETECTION METHOD”, created on Nov. 26, 2012, and having a size of3,640 bytes and is filed concurrently with the specification. Thesequence listing contained in this ASCII formatted document is part ofthe specification and is herein incorporated by reference in itsentirety.

FIELD OF THE INVENTION

The present disclosure relates in part to a method for screening genomicloci of plant events. More particularly, the present disclosure relatesin part to a high throughput method for detecting and identifying plantevents that contain a disruption within a targeted genomic loci. Inaddition, the present disclosure relates in part to a high throughputmethod for detecting and identifying plant events that contain anexogenous donor DNA polynucleotide inserted within a targeted genomicloci. The method is readily applicable for screening plant eventsproduced via a targeting method which results from the use of a sitespecific nuclease.

BACKGROUND OF THE INVENTION

Targeted genome modification of plants has been a long-standing andelusive goal of both applied and basic research. Methods andcompositions to target and cleave genomic DNA by site specific nucleases(Zinc Finger Nucleases (ZFNs), Meganucleases, CRISPRS and TALENS) arebeing developed to reach this goal. The site specific cleavage ofgenomic loci by ZFNs can be used, for example, to induce targetedmutagenesis, induce targeted deletions of cellular DNA sequences, andfacilitate targeted recombination of an exogenous donor DNApolynucleotide within a predetermined genomic locus. See, for example,U.S. Patent Publication No. 20030232410; 20050208489; 20050026157;20050064474; and 20060188987, and International Patent Publication No.WO 2007/014275, the disclosures of which are incorporated by referencein their entireties for all purposes. U.S. Patent Publication No.20080182332 describes use of non-canonical zinc finger nucleases (ZFNs)for targeted modification of plant genomes and U.S. Patent PublicationNo. 20090205083 describes ZFN-mediated targeted modification of a plantEPSPs genomic locus. In addition, Moehle et al. (2007) Proc. Natl. Acad.Sci. USA 104(9): 3055-3060 describe using designed ZFNs for targetedgene addition at a specified genomic locus. Current methods of targetingtypically involve co-transformation of plant tissue with a donor DNApolynucleotide containing at least one transgene and a site specificnuclease (e.g., ZFN) which is designed to bind and cleave a specificgenomic locus. The donor DNA polynucleotide is stably inserted withinthe cleaved genomic locus resulting in targeted gene addition at aspecified genomic locus.

Unfortunately, reported and observed frequencies of targeted genomicmodification indicate that targeting a genomic loci within plants isrelatively inefficient. The reported inefficiency necessitates thescreening of a large number of plant events to identify a specific eventcontaining the targeted genomic loci. Most current reported plant eventanalyses rely on a single analytical method for confirming targeting,which may lead to inaccurate estimation of targeting frequencies and lowconfidence outcomes.

Therefore, there is a need in the art for screening methods, optionallyapplicable as high throughput methods, for the rapid identification ofplant events containing a targeted genomic loci. In addition, astargeted gene insertion occurs in conjunction with random geneinsertion, desirable screening methods would specifically identifytargeting of genomic loci within a background of random insertions.

BRIEF SUMMARY OF THE INVENTION

In an embodiment, the disclosure relates to a method for identifying thepresence of a donor DNA polynucleotide inserted within a targetedgenomic locus comprising amplifying in a first amplification reaction agenomic DNA sample comprising the targeted genomic locus using a firstplurality of oligonucleotides that bind under hybridization conditionsproximal to the targeted genomic locus to thereby generate a firstamplicon comprising the targeted genomic locus, and detecting thepresence or absence of the first amplicon, wherein the absence of thefirst amplicon indicates the presence of the donor DNA polynucleotidewithin the targeted genomic locus.

In a further embodiment, the method comprises amplifying in a secondamplification reaction the genomic DNA sample using a second pluralityof oligonucleotides that bind under hybridization conditions proximal tothe targeted genomic locus and within the donor DNA polynucleotide togenerate a second amplicon comprising at least a portion of the targetedgenomic locus and at least a portion of the donor DNA polynucleotide,and detecting the presence or absence of the second amplicon, whereinthe presence of an amplified product indicates the presence of the donorDNA polynucleotide within the targeted genomic locus.

In yet another embodiment, the method comprises identification of adisruption of a genomic locus from a plurality of plant cells comprisingamplifying in a first amplification reaction a genomic DNA samplecomprising the disrupted genomic locus using a plurality ofoligonucleotides that bind under hybridization conditions proximal tothe disrupted genomic locus to generate a first amplicon comprising thedisrupted genomic locus, quantitating the results of the firstamplification reaction, amplifying in a second amplification reaction agenomic DNA sample comprising the disrupted genomic locus using theplurality of oligonucleotides that bind under hybridization conditionsproximal to the disrupted genomic locus, to thereby generate a secondamplicon comprising the disrupted genomic locus, quantitating theresults of the second amplification reaction, and comparing the quantityof the first and second amplification reactions, wherein the quantity ofthe first amplification reaction comprises a lower quantity of amplifiedproduct as compared to the second amplification reaction therebyindicating the disruption of a genomic locus in the first ampliconsamples.

In another embodiment, the disclosure describes a method for identifyinga disruption of a genomic locus comprising amplifying in a firstamplification reaction a genomic DNA sample comprising the disruptedgenomic locus using a plurality of oligonucleotides that bind underhybridization conditions proximal to the disrupted genomic locus togenerate a first amplicon comprising the disrupted genomic locus, anddetecting the presence or absence of the first amplicon, wherein theabsence of the amplicon indicates the disruption of a genomic locus.

Further embodiments of the method include quantitating the results ofthe first amplification reaction, quantitating the results of the secondamplification reaction, comparing the results of the first and secondamplification reactions, and determining the presence or absence of thedonor DNA polynucleotide within the targeted genomic locus, wherein thedonor DNA polynucleotide is confirmed as inserted within the targetedgenomic locus if the first amplicon is absent and the second amplicon ispresent.

As an embodiment, the first or second amplification reactions are run ina single tube or well in a multiplex format.

In another aspect, an embodiment of the disclosure includes quantitatingthe results of the first and second amplification reactions comprisingproducing a signature profile for one or both of the first and secondamplification reactions. In an exemplary aspect of the embodiment, thesignature profile may be selected from the group consisting of a meltingtemperature curve signature profile and a fluorescence signatureprofile.

Additional embodiments include a signature profile produced from anintercalating DNA dye or a fluorescent dye. Wherein, the intercalatingdye comprises a cyanine dye such as a SYTO13® dye. As an embodiment, theSYTO13® dye is used in an amplification reaction at a concentration ofless than 10 μM, less than 4 μM, or less than 2.7 μM. In additionalembodiments the fluorescent dye is selected from the group consisting ofa HEX fluorescent dye, a FAM fluorescent dye, a JOE fluorescent dye, aTET fluorescent dye, a Cy 3 fluorescent dye, a Cy 3.5 fluorescent dye, aCy 5 fluorescent dye, a Cy 5.5 fluorescent dye, a Cy 7 fluorescent dye,and a ROX fluorescent dye. In an embodiment the first or secondplurality of oligonucleotides, or both, comprise a fluorescent dye.

In embodiments, the present disclosure relates to methods andcompositions for identifying the presence of a donor insertion within atargeted genomic locus, and selecting a transgenic event comprising adonor insertion within a targeted genomic locus. An additionalembodiment includes the plant, comprising the transgenic event.

Further embodiments may comprise a dicot plant, wherein the dicot plantis selected from the group consisting of a soybean plant, a canola plantand a cotton plant. In addition, further embodiments may comprise amonocot plant, wherein the monocot plant is selected from the groupconsisting of a corn plant, a rice plant, and wheat plant.

Additional embodiments include a genomic locus that is cleaved by a sitespecific nuclease. Exemplary site specific nucleases may comprises aZinc Finger Nuclease, a Meganuclease, CRISPR, or a TALEN nuclease or anyother site specific nuclease.

In another aspect, embodiments of the disclosure include anamplification reaction, wherein amplify is completed using a polymerasechain reaction.

Further embodiments of the disclosure include an amplicon comprising a5′ junction and a 3′ junction of the donor DNA polynucleotide and thetargeted genomic locus. In addition, embodiments of the disclosureinclude an amplicon comprising a 5′ junction or a 3′ junction of thedonor DNA polynucleotide and the targeted genomic locus.

Embodiments also include a donor DNA polynucleotide comprising at leastone gene expression cassette.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by study of thefollowing descriptions.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates an analytical process for identifying plant eventscomprising a targeted genomic locus.

FIG. 2 depicts the results of a ZFN quantitative PCR (qPCR) disruptionassay at a targeted locus: screening results of a subset of events. Thearrow indicates a transgenic event with a disrupted locus as evidencedby drop in detectable qPCR signal.

FIG. 3 depicts the screening which is completed with the In-Out PCRamplification process. FIG. 3A depicts a targeted genomic locus(“Locus”) with no transgene insertion. In-Out PCR will result in noamplified product. FIG. 3B depicts the Locus with targeted transgeneinsertion. In-Out PCR will result in a 2 kb amplicon from the 5′ end ofthe locus and a 1.5 kb amplicon from the 3′ end.

FIGS. 4A and 4B depict an In-Out PCR analysis. FIG. 4A depicts afluorescence signal signature profile generated from amplificationreactions using SYTO13® dye (Invitrogen, Carlsbad, Calif.), which can beused to identify positive transgene insertion events. FIG. 4B depictsmelting temperature signature profiles generated from amplificationreactions of samples as compared to a positive control, which can beused to confirm the positive fluorescence signals.

FIG. 5 shows targeted events that were identified using the disruptionassay and the In-Out PCR reaction of the presently disclosed subjectmatter.

FIG. 6 depicts a ZFN disruption assay. Samples shown in the top bracketdo not contain a disrupted genomic locus and samples shown in the bottombracket do contain a disrupted genomic locus.

FIG. 7 depicts another ZFN disruption assay. Samples shown in the topbracket do not contain a disrupted genomic locus and samples shown inthe bottom bracket do contain a disrupted genomic locus.

FIG. 8 depicts an eZFN disruption assay at the ELP locus region. FIG. 8(a) provides an illustration of the genomic fragment containing the ELPwhich was produced by the integration of a T-strand from pDAB 105818.This schematic identifies the relative location of primers and probes(MAS621, MAS622 and probe UPL67) used for the disruption assay. FIG. 8(b) provides the screening results of 354 pat positive events. Adisrupted ELP locus is indicated by drop in detectable qPCR signal.Accordingly samples shown in the top bracket do not contain a disruptedELP genomic locus and samples shown in the bottom bracket do contain adisrupted ELP genomic locus.

FIG. 9 depicts an In-Out PCR analysis. FIG. 9A depicts a meltingtemperature signal profile generated from amplification reactions of the5′ donor/ELP locus junction using SYTO13® dye (Invitrogen, Carlsbad,Calif.), which can be used to identify positive transgene insertionevents. FIG. 9B depicts a melting temperature signal profile generatedfrom amplification reactions of the 3′ donor/ELP locus junction usingSYTO13® dye (Invitrogen, Carlsbad, Calif.), which can be used toidentify positive transgene insertion events.

FIG. 10 provides an illustration of ZF binding sequences withcorresponding zinc fingers and hydrolysis probes for use in the ELPlocus disruption assay: the eZFN8 line represents the probe fordetection of activities from eZFN8 with spacer GTGAGA in between bindingsite sequences SBS15590 and SBS18473; and the eZFN1 line represents theprobe for detection of activities from eZFN1 with spacer GTGGAT inbetween the second set of binding site sequences, SBS15590 and SBS8196.The top sequence is provided as SEQ ID NO:39 and the complementarysequence shown in the bottom of the figure is provided as SEQ ID NO:40.

FIG. 11 illustrates an overlay plot of normalized aad-1 (in circles) andELP copy number (in addition signs). FIG. 11 (a) shows the disruptionassay results of 1125 samples crossed with eZFN1 excisors: 425 with ELPcopy number less than 0.05 (cut), 95 between 0.05 to 0.4 (chimeric).FIG. 11 (b) shows the disruption assay results of 697 samples crossedwith eZFN8 excisors: 488 with ELP copy number less than 0.05, 1 between0.05 to 0.4.

FIG. 12 provides a sequence alignment for ELP genomic locus samples cutwith eZFN1 (pDAB105825) and eZFN8 (pDAB105828). Polynucleotide spacersbetween ZFN recognition sites are indicated in red boxes. Bases shown asdashes (-) are deletions representing a minimum of one missing base ofthe sequence.

DETAILED DESCRIPTION

Novel methods have now been invented for rapid screening, identificationand characterization of site specific nuclease targeted plant events.The methods can be used to analyze the intactness of the genomic targetlocus via a first amplification reaction to determine if the genomictarget locus has been disrupted. Events which are identified to containa disrupted genomic locus can be subsequently screened via a secondamplification reaction to confirm the presence of an exogenous donor DNApolynucleotide within the targeted genomic locus. As such, large numbersof plant events can be analyzed and screened to identify and selectspecific events which have a donor DNA polynucleotide inserted within atargeted genomic locus. The presently disclosed subject matter furtherincludes plants and plant cells comprising nuclease targeted plantevents selected utilizing the novel screening methods.

Demonstrated herein are novel methods for screening plant events for thedisruption of a genomic locus, which is a result of genomic DNA cleavageby a site specific nuclease. The disrupted genomic locus may comprisethe presence of a donor DNA polynucleotide, or the disrupted genomiclocus may comprise insertions and/or deletions (also described asInDels). The methods utilize two initial amplification reactions as ascreening assay. The first amplification reaction is a disruption assay,wherein the presence of a donor DNA polynucleotide inserted within atargeted genomic locus is identified by an amplification reaction inwhich the absence of an amplicon indicates that a donor DNApolynucleotide is present within the targeted genomic locus. The secondamplification reaction is an “In-Out” PCR amplification reaction forscreening the 3′ and/or the 5′ junction sequences of a donor DNApolynucleotide targeted genomic locus. The presence of an amplifiedproduct which contains the 3′ and/or 5′ junction sequence indicates thatthe donor DNA polynucleotide is present within the targeted genomiclocus.

By deploying two distinct amplification reaction screening assays, thedisruption amplification reaction and the In-Out amplification reaction,the probability of identifying a positive target event from the largenumber of non-targeted events is greatly increased. The disruptionamplification reaction was designed to allow for rapid analysis of alarge number of samples in a high throughput manner. Furthermore, thisassay can identify and characterize events for both donor DNApolynucleotide insertion or ZFN cleavage within a targeted genomiclocus. The In-Out amplification reaction provides an alternativeanalytical approach. This assay is used to identify the presence of the3′ and 5′ junctions to confirm the presence of a donor DNApolynucleotide inserted within a targeted genomic locus. The In-Outamplification reaction can be used to confirm that the targeted eventsidentified in the disruption assay actually contain a complete,full-length targeted insertion within a genomic locus. When thedisruption amplification reaction is run in conjunction with the In-Outamplification reaction, the compiled data can be analyzed to determinethe limiting factors for targeting of a donor DNA polynucleotide withina genomic locus (e.g., ZFN cleavage or donor insertion as a limitingfactor for producing events containing a donor DNA polynucleotide withina targeted genomic locus). Utilizing two different screening assays withvarying methodology increases the likelihood of finding a targeted eventwhich contains a donor DNA polynucleotide within a targeted genomiclocus. Moreover, the disclosed methods can be deployed as highthroughput assays allowing for the rapid and efficient identification ofa subset of samples that can then be further analyzed by other molecularconfirmation methods. The disclosed screening assays describe highquality, high throughput processes for identifying and obtainingtargeted transgene insertion events. Furthermore, the methodology isreadily applicable for the analysis of any plant species.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure relates. In case of conflict, thepresent application including the definitions will control. Unlessotherwise required by context, singular terms shall include pluralitiesand plural terms shall include the singular. All publications, patentsand other references mentioned herein are incorporated by reference intheir entireties for all purposes as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference, unless only specific sections of patents orpatent publications are indicated to be incorporated by reference.

In order to further clarify this disclosure, the following terms,abbreviations and definitions are provided.

As used herein, the terms ““comprises”,” ““comprising”,” ““includes”,”““including”,” ““has”,” ““having”,” ““contains”,” or ““containing”,” orany other variation thereof, are intended to be non-exclusive oropen-ended. For example, a composition, a mixture, a process, a method,an article, or an apparatus that comprises a list of elements is notnecessarily limited to only those elements but may include otherelements not expressly listed or inherent to such composition, mixture,process, method, article, or apparatus. Further, unless expressly statedto the contrary, ““or”” refers to an inclusive or and not to anexclusive or. For example, a condition A or B is satisfied by any one ofthe following: A is true (or present) and B is false (or not present), Ais false (or not present) and B is true (or present), and both A and Bare true (or present).

Also, the indefinite articles ““a”” and ““an”” preceding an element orcomponent of an embodiment of the disclosure are intended to benonrestrictive regarding the number of instances, i.e., occurrences ofthe element or component. Therefore ““a”” or ““an”” should be read toinclude one or at least one, and the singular word form of the elementor component also includes the plural unless the number is obviouslymeant to be singular.

The term ““invention”” or ““present invention”” as used herein is anon-limiting term and is not intended to refer to any single embodimentof the particular invention but encompasses all possible embodiments asdisclosed in the application.

The term “plant” as used herein includes, but is not limited to, anydescendant, cell, tissue, or part of a plant.

Described herein are methods of identifying the presence of a donor DNApolynucleotide inserted within a targeted genomic loci. DNApolynucleotides for insertion can also be referred to, and are intendedto include, as “exogenous” polynucleotides, “donor” polynucleotides or“molecules” or “transgenes.”

In certain embodiments, the donor DNA polynucleotide includes sequences(e.g., coding sequences, also referred to as transgenes) greater than 1kb in length, for example between 2 and 200 kb, between 2 and 10 kb (orany value therebetween). The donor DNA polynucleotide can also includeat least one site specific nuclease target site, for example at leastone ZFN, Meganuclease, CRISPR, or TALEN target site may be included inthe donor DNA polynucleotide. The donor DNA polynucleotide can includeat least 1 target site, for example for a pair of ZFNs, CRISPRs, orTALENs to recognize and bind and cleave. Typically, the nuclease targetsites are outside the transgene sequences, for example, 5′ or 3′ to thetransgene sequences, for cleavage and removal of the interveningtransgene, if desired. The nuclease cleavage site(s) may be for anynuclease(s). In certain embodiments, the nuclease target site(s)contained in the double-stranded donor DNA polynucleotide are for thesame nuclease(s) used to cleave the endogenous genomic target into whichthe donor DNA polynucleotide is inserted.

The transgenes comprised within the donor DNA polynucleotide sequencesdescribed herein may be isolated from plasmids, cells or other sourcesusing standard techniques known in the art such as PCR. Donor DNApolynucleotide sequences for use can include varying types of topology,including circular supercoiled, circular relaxed, linear and the like.Alternatively, they may be chemically synthesized using standardoligonucleotide synthesis techniques. In addition, donor DNApolynucleotide sequences may be methylated or lack methylation. DonorDNA polynucleotide sequences may be in the form of bacterial or yeastartificial chromosomes (BACs or YACs), or as plasmid vectors. Donor DNApolynucleotide sequences may be from a T-strand which is introduced intoa plant cell by Agrobacterium tumefaciens.

The double-stranded donor DNA polynucleotides described herein mayinclude one or more non-natural bases and/or backbones. In particular,insertion of a donor DNA polynucleotide sequence with methylatedcytosines may be carried out using the methods described herein toachieve a state of transcriptional quiescence in a region of interest.

The donor DNA polynucleotide may comprise any exogenous sequence ofinterest. Exemplary donor DNA polynucleotide sequences include, but arenot limited to any polypeptide coding sequence (e.g., cDNAs), promotersequences, enhancer sequences, epitope tags, marker genes, cleavageenzyme recognition sites and various types of expression constructs.Marker genes include, but are not limited to, sequences encodingproteins that mediate herbicide resistance (e.g., HPPD resistance, 2,4-Dresistance, glufosinate resistance, or glyphosate resistance, inaddition to other know herbicide resistance proteins), sequencesencoding colored or fluorescent or luminescent proteins (e.g., greenfluorescent protein, enhanced green fluorescent protein, red fluorescentprotein, luciferase), and proteins which mediate enhanced cell growthand/or gene amplification (e.g., dihydrofolate reductase). Epitope tagsinclude, for example, one or more copies of FLAG, HIS, MYC, TAP, HA orany detectable amino acid sequence.

The terms “polynucleotide,” “nucleic acid,” and “nucleic acid molecule”are intended to encompass a singular nucleic acid as well as pluralnucleic acids, a nucleic acid fragment, variant, or derivative thereof,or construct, e.g., messenger RNA (mRNA) or plasmid DNA (pDNA). Apolynucleotide or nucleic acid can contain the nucleotide sequence ofthe full-length cDNA sequence, or a fragment thereof, including theuntranslated 5′ and 3′ sequences and the coding sequences. Unlessspecified otherwise, a polynucleotide or nucleic acid can be composed ofany polyribonucleotide or polydeoxyribonucleotide, which may beunmodified RNA or DNA or modified RNA or DNA. For example, apolynucleotide or nucleic acid can be composed of single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions.These terms also embrace chemically, enzymatically, or metabolicallymodified forms of a polynucleotide or nucleic acid.

A polynucleotide or nucleic acid sequence can be referred to as“isolated,” in which it has been removed from its native environment.For example, a heterologous polynucleotide or nucleic acid encoding apolypeptide or polypeptide fragment having glyphosate tolerance activitycontained in a vector is considered isolated for the purposes of thepresent disclosure. Further examples of an isolated polynucleotide ornucleic acid include recombinant polynucleotide maintained inheterologous host cells or a purified (partially or substantially)polynucleotide or nucleic acid in solution. An isolated polynucleotideor nucleic acid according to embodiments of the present disclosurefurther includes such molecules produced synthetically. An isolatedpolynucleotide or nucleic acid in the form of a polymer of DNA may becomprised of one or more segments of cDNA, genomic DNA or synthetic DNA.

The term “gene” refers to a nucleic acid sequence encodes functionalproduct molecules, either RNA or protein, optionally includingregulatory sequences preceding (5′ non-coding sequences) and following(3′ non-coding sequences) the coding sequence.

As used herein the term “coding region” refers to a DNA sequence thatcodes for a specific amino acid sequence. “Suitable regulatorysequences” refer to nucleotide sequences located upstream (5′ non-codingsequences), within, or downstream (3′ non-coding sequences) of a codingsequence, and which influence the transcription, RNA processing orstability, or translation of the associated coding sequence. Regulatorysequences can include promoters, translation leader sequences, introns,polyadenylation recognition sequences, RNA processing site, effectorbinding site and stem-loop structure.

As used herein, the term “polypeptide” is intended to encompass asingular “polypeptide” as well as plural “polypeptides” and fragmentsthereof, and refers to a molecule composed of monomers (amino acids)linearly linked by amide bonds (also known as peptide bonds). The term“polypeptide” refers to any chain or chains of two or more amino acids,and does not refer to a specific length of the product. Thus, peptides,dipeptides, tripeptides, oligopeptides, “protein,” “amino acid chain,”or any other term used to refer to a chain or chains of two or moreamino acids, are included within the definition of “polypeptide,” andthe term “polypeptide” can be used instead of, or interchangeably withany of these terms. A polypeptide can be derived from a naturalbiological source or produced by recombinant technology, but is notnecessarily translated from a designated nucleic acid sequence. It canbe generated in any manner, including by chemical synthesis.

By an “isolated” polypeptide or a fragment, variant, or derivativethereof is intended a polypeptide that is not in its natural milieu. Noparticular level of purification is required. Thus, reference to“isolated” signifies the involvement of the “hand of man” as describedherein. For example, an isolated polypeptide can be removed from itsnative or natural environment. Recombinant polypeptides and proteinsexpressed in host cells are considered isolated for purposes of thedisclosure, as are native or recombinant polypeptides which have beenseparated, fractionated, or partially or substantially purified by anysuitable technique.

As used herein, “native” refers to the form of a polynucleotide, gene orpolypeptide as found in nature with its own regulatory sequences, ifpresent.

As used herein, “endogenous” refers to the native form of apolynucleotide, gene or polypeptide in its natural location in theorganism or in the genome of an organism. “Endogenous polynucleotide”includes a native polynucleotide in its natural location in the genomeof an organism. “Endogenous gene” includes a native gene in its naturallocation in the genome of an organism. “Endogenous polypeptide” includesa native polypeptide in its natural location in the organism.

As used herein, “heterologous” refers to a polynucleotide, gene orpolypeptide not normally found in the host organism but that isintroduced into the host organism. “Heterologous polynucleotide”includes a native coding region, or portion thereof, that isreintroduced into the source organism in a form that is different fromthe corresponding native polynucleotide. “Heterologous gene” includes anative coding region, or portion thereof, that is reintroduced into thesource organism in a form that is different from the correspondingnative gene. For example, a heterologous gene may include a nativecoding region that is a portion of a chimeric gene including non-nativeregulatory regions that is reintroduced into the native host.“Heterologous polypeptide” includes a native polypeptide that isreintroduced into the source organism in a form that is different fromthe corresponding native polypeptide. The subject genes and proteins canbe fused to other genes and proteins to produce chimeric or fusionproteins. The genes and proteins useful in accordance with embodimentsof the subject disclosure include not only the specifically exemplifiedfull-length sequences, but also portions, segments and/or fragments(including contiguous fragments and internal and/or terminal deletionscompared to the full-length molecules) of these sequences, variants,mutants, chimerics, and fusions thereof.

In an embodiment, the donor DNA polynucleotide comprises apolynucleotide encoding any polypeptide of which expression in the cellis desired, including, but not limited to a gene expression cassettecomprising promoters, 3′ UTR's, herbicide resistance traits, insectresistance traits, modified oil traits, agronomic traits, and functionalfragments of any of the above. The coding sequences may be, for example,cDNAs.

The term “promoter” refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. Promoters can be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or evencomprise synthetic DNA segments. It is understood by those skilled inthe art that different promoters can direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental or physiological conditions.Promoters which cause a gene to be expressed in most cell types at mosttimes are commonly referred to as “constitutive promoters.” It isfurther recognized that since in most cases the exact boundaries ofregulatory sequences have not been completely defined, DNA fragments ofdifferent lengths may have identical promoter activity.

The term “operably linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis affected by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of effecting the expression ofthat coding sequence (e.g., that the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in sense or antisenseorientation.

The term “expression,” as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid fragment of embodiments of the disclosure. Expression mayalso refer to translation of mRNA into a polypeptide.

The term “overexpression” as used herein, refers to expression that ishigher than endogenous expression of the same or related gene. Aheterologous gene is overexpressed if its expression is higher than thatof a comparable endogenous gene.

As used herein the term “transformation” refers to the transfer andintegration of a nucleic acid or fragment into a host organism,resulting in genetically stable inheritance. Host organisms containingthe transformed nucleic acid fragments are referred to as “transgenic”or “recombinant” or “transformed” organisms. Known methods oftransformation include Agrobacterium tumefaciens- or Agrobacteriumrhizogenes-mediated transformation, calcium phosphate transformation,polybrene transformation, protoplast fusion, electroporation, ultrasonicmethods (e.g., sonoporation), liposome transformation, microinjection,naked DNA, plasmid vectors, viral vectors, biolistics (microparticlebombardment), silicon carbide WHISKERS™ mediated transformation, aerosolbeaming, or PEG transformation as well as other possible methods.

The terms “plasmid” and “vector” as used herein, refer to an extrachromosomal element often carrying genes which are not part of thecentral metabolism of the cell, and usually in the form of circulardouble-stranded DNA molecules. Such elements may be autonomouslyreplicating sequences, genome integrating sequences, phage or nucleotidesequences, linear or circular, of a single- or double-stranded DNA orRNA, derived from any source, in which a number of nucleotide sequenceshave been joined or recombined into a unique construction which iscapable of introducing a promoter fragment and DNA sequence for aselected gene product along with appropriate 3′ untranslated sequenceinto a cell.

As used herein the term “codon degeneracy” refers to the nature in thegenetic code permitting variation of the nucleotide sequence withoutaffecting the amino acid sequence of an encoded polypeptide. The skilledartisan is well aware of the “codon-bias” exhibited by a specific hostcell in usage of nucleotide codons to specify a given amino acid.Therefore, when synthesizing a gene for improved expression in a hostcell, it is desirable to design the gene such that its frequency ofcodon usage approaches the frequency of preferred codon usage of thehost cell.

The term “codon-optimized” as it refers to genes or coding regions ofnucleic acid molecules for transformation of various hosts, refers tothe alteration of codons in the gene or coding regions of the nucleicacid molecules to reflect the typical codon usage of the host organismwithout altering the polypeptide encoded by the DNA. Such optimizationincludes replacing at least one, or more than one, or a significantnumber, of codons with one or more codons that are more frequently usedin the genes of that organism.

The term “percent identity” (or “% identity”), as known in the art, is arelationship between two or more polypeptide sequences or two or morepolynucleotide sequences, as determined by comparing the sequences. Inthe art, “identity” also means the degree of sequence relatednessbetween polypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”and “similarity” can be readily calculated by known methods, includingbut not limited to those disclosed in: 1.) Computational MolecularBiology (Lesk, A. M., Ed.) Oxford University: NY (1988); 2.)Biocomputing: Informatics and Genome Projects (Smith, D. W., Ed.)Academic: NY (1993); 3.) Computer Analysis of Sequence Data, Part I(Griffin, A. M., and Griffin, H. G., Eds.) Humania: NJ (1994); 4.)Sequence Analysis in Molecular Biology (von Heinje, G., Ed.) Academic(1987); and 5.) Sequence Analysis Primer (Gribskov, M. and Devereux, J.,Eds.) Stockton: NY (1991).

Techniques for determining nucleic acid and amino acid sequence identityare known in the art. Typically, such techniques include determining thenucleotide sequence of the mRNA for a gene and/or determining the aminoacid sequence encoded thereby, and comparing these sequences to a secondnucleotide or amino acid sequence. Genomic sequences can also bedetermined and compared in this fashion. In general, identity refers toan exact nucleotide-to-nucleotide or amino acid-to-amino acidcorrespondence of two polynucleotides or polypeptide sequences,respectively. Two or more sequences (polynucleotide or amino acid) canbe compared by determining their percent identity. The percent identityof two sequences, whether nucleic acid or amino acid sequences, is thenumber of exact matches between two aligned sequences divided by thelength of the shorter sequences and multiplied by 100. See Russell, R.,and Barton, G., “Structural Features can be Unconserved in Proteins withSimilar Folds,” J. Mol. Biol. 244, 332-350 (1994), at p. 337, which isincorporated herein by reference in its entirety.

In addition, methods to determine identity and similarity are codifiedin publicly available computer programs. Sequence alignments and percentidentity calculations can be performed, for example, using the AlignXprogram of the VECTOR NTI® suite (Invitrogen, Carlsbad, Calif.) orMEGALIGN™ program of the LASERGENE™ bioinformatics computing suite(DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences isperformed using the “Clustal method of alignment” which encompassesseveral varieties of the algorithm including the “Clustal V method ofalignment” corresponding to the alignment method labeled Clustal V(disclosed by Higgins and Sharp, CABIOS. 5:151-153 (1989); Higgins, D.G. et al., Comput. Appl. Biosci., 8:189-191 (1992)) and found in theMEGALIGN™ program of the LASERGENE™ bioinformatics computing suite(DNASTAR Inc.). For multiple alignments, the default values correspondto GAP PENALTY=10 and GAP LENGTH PENALTY=10. Default parameters forpairwise alignments and calculation of percent identity of proteinsequences using the Clustal method are KTUPLE=1, GAP PENALTY=3, WINDOW=5and DIAGONALS SAVED=5. For nucleic acids these parameters are KTUPLE=2,GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4. After alignment of thesequences using the Clustal V program, it is possible to obtain a“percent identity” by viewing the “sequence distances” table in the sameprogram. Additionally the “Clustal W method of alignment” is availableand corresponds to the alignment method labeled Clustal W (described byHiggins and Sharp, CABIOS. 5:151-153 (1989); Higgins, D. G. et al.,Comput. Appl. Biosci. 8:189-191(1992)) and found in the MEGALIGN™ v6.1program of the LASERGENE™ bioinformatics computing suite (DNASTAR Inc.).Default parameters for multiple alignment (GAP PENALTY=10, GAP LENGTHPENALTY=0.2, Delay Divergen Seqs(%)=30, DNA Transition Weight=0.5,Protein Weight Matrix=Gonnet Series, DNA Weight Matrix=IUB). Afteralignment of the sequences using the Clustal W program, it is possibleto obtain a “percent identity” by viewing the “sequence distances” tablein the same program.

The nucleic acid probes and primers of embodiments of the presentdisclosure can hybridize under hybridization conditions to a target DNAsequence within an amplification reaction. Any conventional nucleic acidhybridization or amplification method can be used to identify thepresence of donor DNA polynucleotide inserted within a targeted genomiclocus. Nucleic acid molecules, oligonucleotides or fragments thereof arecapable of specifically hybridizing to other nucleic acid moleculesunder certain conditions. As used herein, two nucleic acid molecules aresaid to be capable of specifically hybridizing to one another if the twomolecules are capable of forming an anti-parallel, double-strandednucleic acid structure. A nucleic acid molecule is said to be the“complement” of another nucleic acid molecule if the two nucleic acidmolecules exhibit complete complementarity. As used herein, moleculesare said to exhibit “complete complementarity” when every nucleotide ofone of the molecules is complementary to a nucleotide of the other.Molecules that exhibit complete complementarity will generally hybridizeto one another with sufficient stability to permit them to remainannealed to one another under conventional “high-stringency” conditions.Conventional high-stringency conditions are described by Sambrook etal., 1989.

Two molecules are said to exhibit “minimal complementarity” if they canhybridize to one another with sufficient stability to permit them toremain annealed to one another under at least conventional“low-stringency” conditions. Conventional low-stringency conditions aredescribed by Sambrook et al., 1989. In order for a nucleic acid moleculeto serve as a primer or probe, it need only exhibit the minimalcomplementarity of sequence to be able to form a stable double-strandedstructure under the particular solvent and salt concentrations employed.

Factors that affect the stringency of hybridization are well-known tothose of skill in the art and include, but are not limited to,temperature, pH, ionic strength, and concentration of organic solventssuch as, for example, formamide and dimethyl sulfoxide. As is known tothose of skill in the art, hybridization stringency is increased byhigher temperatures, lower ionic strength and lower solventconcentrations.

The term “stringent condition” or “stringency conditions” isfunctionally defined with regard to the hybridization of a nucleic-acidprobe to a target nucleic acid (i.e., to a particular nucleic-acidsequence of interest) by the specific hybridization procedure discussedin Sambrook et al., 1989.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na⁺ ion, typically about 0.01 to1.0 M Na⁺ ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1.0 M NaCl, 0.1% SDS (sodium dodecyl sulfate) at 37°C., and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodiumcitrate) at 50 to 55° C. Exemplary moderate stringency conditionsinclude hybridization in 40 to 45% formamide, 1.0 M NaCl, 0.1% SDS at37° C., and a wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary highstringency conditions include hybridization in 50% formamide, 1.0 MNaCl, 0.1% SDS at 37° C., and a wash in 0.1×SSC at 60 to 65° C.

Specificity is typically a function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the T_(m) can be approximated fromthe equation T_(m)=81.5° C.+16.6 (log M)+0.41(% GC)−0.61(% form.)−500/L,where M is the molarity of monovalent cations, % GC is the percentage ofguanosine and cytosine nucleotides in the DNA, % form. is the percentageof formamide in the hybridization solution, and L is the length of thehybrid in base pairs (Meinkoth and Wahl, 1984). The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of acomplementary target sequence hybridizes to a perfectly matched probe.T_(m) is reduced by about 1° C. for each 1% of mismatching; thus, T_(m),hybridization, and/or wash conditions can be adjusted for sequences ofthe desired identity to hybridize. For example, if sequences with 90%identity are sought, the T_(m) can be decreased 10° C. Generally,stringent conditions are selected to be about 5° C. lower than thethermal melting point (T_(m)) for the specific sequence and itscomplement at a defined ionic strength and pH. However, severelystringent conditions can utilize a hybridization and/or wash at 1, 2, 3,or 4° C. lower than the thermal melting point (T_(m)); moderatelystringent conditions can utilize a hybridization and/or wash at 6, 7, 8,9, or 10° C. lower than the thermal melting point (T_(m)); lowstringency conditions can utilize a hybridization and/or wash at 11 to20° C. lower than the thermal melting point (T_(m)). Using the equation,hybridization and wash compositions, and desired T_(m), those ofordinary skill will understand that variations in the stringency ofhybridization and/or wash solutions are inherently described. If thedesired degree of mismatching results in a T_(m) of less than 45° C.(aqueous solution) or 32° C. (formamide solution), it is preferred toincrease the SSC concentration so that a higher temperature can be used.An extensive guide to the hybridization of nucleic acids is found (1997)Ausubel et al., Short Protocols in Molecular Biology, pages 2-40, 3^(rd)Ed. (1997) and Sambrook et al. (1989).

In an embodiment, the subject disclosure relates to the introduction ofa donor DNA polynucleotide which is inserted within a targeted genomelocus. Standard recombinant DNA and molecular cloning techniques for theconstruction of a donor DNA polynucleotide that as an embodimentcomprises a gene expression cassette as used here are well known in theart and are described, e.g., by Sambrook et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (1989); and by Silhavy et al., Experiments withGene Fusions, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y. (1984); and by Ausubel et al., Current Protocols in MolecularBiology, published by Greene Publishing Assoc. and Wiley-Interscience(1987).

In methods disclosed herein, a number of promoters that directexpression of a gene in a plant can be employed. Such promoters can beselected from constitutive, chemically-regulated, inducible,tissue-specific, and seed-preferred promoters. The promoter used todirect expression of a nucleic acid depends on the particularapplication. For example, a strong constitutive promoter suited to thehost cell is typically used for expression and purification of expressedproteins.

Non-limiting examples of preferred plant promoters include promotersequences derived from A. thaliana ubiquitin-10 (ubi-10) (Callis, etal., 1990, J. Biol. Chem., 265:12486-12493); A. tumefaciens mannopinesynthase (Δmas) (Petolino et al., U.S. Pat. No. 6,730,824); and/orCassava Vein Mosaic Virus (CsVMV) (Verdaguer et al., 1996, PlantMolecular Biology 31:1129-1139). Other constitutive promoters include,for example, the core Cauliflower Mosaic Virus 35S promoter (Odell etal. (1985) Nature 313:810-812); Rice Actin promoter (McElroy et al.(1990) Plant Cell 2:163-171); Maize Ubiquitin promoter (U.S. Pat. No.5,510,474; Christensen et al. (1989) Plant Mol. Biol. 12:619-632 andChristensen et al. (1992) Plant Mol. Biol. 18:675-689); pEMU promoter(Last et al. (1991) Theor. Appl. Genet. 81:581-588); ALS promoter (U.S.Pat. No. 5,659,026); Maize Histone promoter (Chabouté et al. PlantMolecular Biology, 8:179-191 (1987)); and the like.

Other applicable plant promoters include tissue specific and induciblepromoters. An inducible promoter is one that is capable of directly orindirectly activating transcription of one or more DNA sequences orgenes in response to an inducer. In the absence of an inducer the DNAsequences or genes will not be transcribed. Typically, the proteinfactor that binds specifically to an inducible regulatory element toactivate transcription is present in an inactive form which is thendirectly or indirectly converted to the active form by the inducer. Theinducer can be a chemical agent such as a protein, metabolite, growthregulator, herbicide or phenolic compound or a physiological stressimposed directly by heat, cold, salt, or toxic elements or indirectlythrough the action of a pathogen or disease agent such as a virus.Typically the protein factor that binds specifically to an inducibleregulatory element to activate transcription is present in an inactiveform which is then directly or indirectly converted to the active formby the inducer. The inducer can be a chemical agent such as a protein,metabolite, growth regulator, herbicide or phenolic compound or aphysiological stress imposed directly by heat, cold, salt, or toxicelements or indirectly through the action of a pathogen or disease agentsuch as a virus. A plant cell containing an inducible regulatory elementmay be exposed to an inducer by externally applying the inducer to thecell or plant such as by spraying, watering, heating or similar methods.

Any inducible promoter can be used in embodiments of the instantdisclosure. See Ward et al., Plant Mol. Biol. 22: 361-366 (1993).Exemplary inducible promoters include ecdysone receptor promoters (U.S.Pat. No. 6,504,082); promoters from the ACE1 system which respond tocopper (Mett et al., Proc. Natl. Acad. Sci. 90: 4567-4571 (1993)); In2-1and In2-2 gene from maize which respond to benzenesulfonamide herbicidesafeners (U.S. Pat. No. 5,364,780; Hershey et al., Mol. Gen. Genetics227: 229-237 (1991) and Gatz et al., Mol. Gen. Genetics 243: 32-38(1994)); Tet repressor from Tn10 (Gatz et al., Mol. Gen. Genet. 227:229-237 (1991); or promoters from a steroid hormone gene, thetranscriptional activity of which is induced by a glucocorticosteroidhormone, Schena et al., Proc. Natl. Acad. Sci. U.S.A. 88: 10421 (1991)and McNellis et al., (1998) Plant J. 14(2):247-257; the maize GSTpromoter, which is activated by hydrophobic electrophilic compounds thatare used as pre-emergent herbicides (see U.S. Pat. No. 5,965,387 andInternational Patent Application, Publication No. WO 93/001294); and thetobacco PR-la promoter, which is activated by salicylic acid (see Ono S,Kusama M, Ogura R, Hiratsuka K., “Evaluation of the Use of the TobaccoPR-la Promoter to Monitor Defense Gene Expression by the LuciferaseBioluminescence Reporter System,” Biosci Biotechnol Biochem. 2011 Sep.23; 75(9):1796-800). Other chemical-regulated promoters of interestinclude tetracycline-inducible and tetracycline-repressible promoters(see, for example, Gatz et al., (1991) Mol. Gen. Genet. 227:229-237, andU.S. Pat. Nos. 5,814,618 and 5,789,156).

Other regulatable promoters of interest include a cold responsiveregulatory element or a heat shock regulatory element, the transcriptionof which can be effected in response to exposure to cold or heat,respectively (Takahashi et al., Plant Physiol. 99:383-390, 1992); thepromoter of the alcohol dehydrogenase gene (Gerlach et al., PNAS USA79:2981-2985 (1982); Walker et al., PNAS 84(19):6624-6628 (1987)),inducible by anaerobic conditions; and the light-inducible promoterderived from the pea rbcS gene or pea psaDb gene (Yamamoto et al.,(1997) Plant J. 12(2):255-265); a light-inducible regulatory element(Feinbaum et al., Mol. Gen. Genet. 226:449, 1991; Lam and Chua, Science248:471, 1990; Matsuoka et al. (1993) Proc. Natl. Acad. Sci. USA90(20):9586-9590; Orozco et al. (1993) Plant Mol. Bio. 23(6):1129-1138),a plant hormone inducible regulatory element (Yamaguchi-Shinozaki etal., Plant Mol. Biol. 15:905, 1990; Kares et al., Plant Mol. Biol.15:225, 1990), and the like. An inducible regulatory element also can bethe promoter of the maize In2-1 or In2-2 gene, which responds tobenzenesulfonamide herbicide safeners (Hershey et al., Mol. Gen. Gene.227:229-237, 1991; Gatz et al., Mol. Gen. Genet. 243:32-38, 1994), andthe Tet repressor of transposon Tn10 (Gatz et al., Mol. Gen. Genet.227:229-237, 1991). Stress inducible promoters include salt/waterstress-inducible promoters such as PSCS (Zang et al., (1997) PlantSciences 129:81-89); cold-inducible promoters, such as, cor15a (Hajelaet al., (1990) Plant Physiol. 93:1246-1252), cor15b (Wilhelm et al.,(1993) Plant Mol Biol 23:1073-1077), wsc1 (Ouellet et al., (1998) FEBSLett. 423-324-328), ci7 (Kirch et al., (1997) Plant Mol Biol.33:897-909), ci21A (Schneider et al., (1997) Plant Physiol. 113:335-45);drought-inducible promoters, such as Trg-31 (Chaudhary et al., (1996)Plant Mol. Biol. 30:1247-57), rd29 (Kasuga et al., (1999) NatureBiotechnology 18:287-291); osmotic inducible promoters, such as Rab17(Vilardell et al., (1991) Plant Mol. Biol. 17:985-93) and osmotin(Raghothama et al., (1993) Plant Mol Biol 23:1117-28); and heatinducible promoters, such as heat shock proteins (Banos et al., (1992)Plant Mol. 19:665-75; Marrs et al., (1993) Dev. Genet. 14:27-41), smHSP(Waters et al., (1996) J. Experimental Botany 47:325-338), and theheat-shock inducible element from the parsley ubiquitin promoter (WO03/102198). Other stress-inducible promoters include rip2 (U.S. Pat. No.5,332,808 and U.S. Publication No. 2003/0217393) and rd29a(Yamaguchi-Shinozaki et al., (1993) Mol. Gen. Genetics 236:331-340).Certain promoters are inducible by wounding, including the AgrobacteriumpMAS promoter (Guevara-Garcia et al., (1993) Plant J. 4(3):495-505) andthe Agrobacterium ORF13 promoter (Hansen et al., (1997) Mol. Gen. Genet.254(3):337-343).

Tissue-preferred promoters can be utilized to target enhancedtranscription and/or expression within a particular plant tissue. Whenreferring to preferential expression, what is meant is expression at ahigher level in the particular plant tissue than in other plant tissue.Examples of these types of promoters include seed preferred expressionsuch as that provided by the phaseolin promoter (Bustos et al., (1989)The Plant Cell Vol. 1, 839-853), and the maize globulin-1 gene(Belanger, et al. (1991) Genetics 129:863-972). For dicots,seed-preferred promoters include, but are not limited to, beanβ-phaseolin, napin, β-conglycinin, soybean lectin, cruciferin, and thelike. For monocots, seed-preferred promoters include, but are notlimited to, maize 15 kDa zein, 22 kDa zein, 27 kDa zein, γ-zein, waxy,shrunken 1, shrunken 2, globulin 1, etc. Seed-preferred promoters alsoinclude those promoters that direct gene expression predominantly tospecific tissues within the seed such as, for example, theendosperm-preferred promoter of γ-zein, the cryptic promoter fromtobacco (Fobert et al., (1994) T-DNA tagging of a seed coat-specificcryptic promoter in tobacco. Plant J. 4: 567-577), the P-gene promoterfrom corn (Chopra et al., (1996) Alleles of the maize P gene withdistinct tissue specificities encode Myb-homologous proteins withC-terminal replacements. Plant Cell 7:1149-1158, Erratum in Plant Cell.1997, 1:109), the globulin-1 promoter from corn (Belenger and Kriz(1991) Molecular basis for Allelic Polymorphism of the maize Globulin-1gene. Genetics 129: 863-972), and promoters that direct expression tothe seed coat or hull of corn kernels, for example the pericarp-specificglutamine synthetase promoter (Muhitch et al., (2002) Isolation of aPromoter Sequence From the Glutamine Synthetase₁₋₂ Gene Capable ofConferring Tissue-Specific Gene Expression in Transgenic Maize. PlantScience 163:865-872).

In addition to the promoter, the expression vector typically contains atranscription unit or expression cassette that contains all theadditional elements required for the expression of the nucleic acid inhost cells, either prokaryotic or eukaryotic. A typical expressioncassette thus contains a promoter operably linked, e.g., to a nucleicacid sequence encoding the protein, and signals required, e.g., forefficient polyadenylation of the transcript, transcriptionaltermination, ribosome binding sites, or translation termination.Additional elements of the cassette may include, e.g., enhancers andheterologous splicing signals.

Other components of the vector may be included, also depending uponintended use of the gene. Examples include selectable markers, targetingor regulatory sequences, transit peptide sequences such as the optimizedtransit peptide sequence (see U.S. Pat. No. 5,510,471) stabilizingsequences such as RB7 MAR (see Thompson and Myatt, (1997) Plant Mol.Biol., 34: 687-692 and International Patent Publication No. WO9727207)or leader sequences, introns etc. General descriptions and examples ofplant expression vectors and reporter genes can be found in Gruber, etal., “Vectors for Plant Transformation” in Methods in Plant MolecularBiology and Biotechnology, Glick et al eds; CRC Press pp. 89-119 (1993).The selection of an appropriate expression vector will depend upon thehost and the method of introducing the expression vector into the host.The expression cassette will also include at the 3′ terminus of theheterologous nucleotide sequence of interest, a transcriptional andtranslational termination region functional in plants. The terminationregion can be native with the promoter nucleotide sequence ofembodiments of the present disclosure, can be native with the DNAsequence of interest, or can be derived from another source. Convenienttermination regions are available from the Ti-plasmid of A. tumefaciens,such as the octopine synthase and nopaline synthase (nos) terminationregions (Depicker et al., Mol. and Appl. Genet. 1:561-573 (1982) andShaw et al. (1984) Nucleic Acids Research vol. 12, No. 20 pp7831-7846(nos)); see also Guerineau et al. Mol. Gen. Genet. 262:141-144(1991); Proudfoot, Cell 64:671-674 (1991); Sanfacon et al. Genes Dev.5:141-149 (1991); Mogen et al. Plant Cell 2:1261-1272 (1990); Munroe etal. Gene 91:151-158 (1990); Ballas et al., Nucleic Acids Res.17:7891-7903 (1989); Joshi et al. Nucleic Acid Res. 15:9627-9639 (1987).

The expression cassettes can additionally contain 5′ leader sequences.Such leader sequences can act to enhance translation. Translationleaders are known in the art and include by way of example, picornavirusleaders, EMCV leader (Encephalomyocarditis 5′ noncoding region),Elroy-Stein et al., Proc. Nat. Acad. Sci. USA 86:6126-6130 (1989);potyvirus leaders, for example, TEV leader (Tobacco Etch Virus)Carrington and Freed Journal of Virology, 64:1590-1597 (1990), MDMVleader (Maize Dwarf Mosaic Virus), Allison et al., Virology 154:9-20(1986); human immunoglobulin heavy-chain binding protein (BiP), Macejaket al., Nature 353:90-94 (1991); untranslated leader from the coatprotein mRNA of alfalfa mosaic virus (AMV RNA 4), Jobling et al., Nature325:622-625 (1987); Tobacco mosaic virus leader (TMV), Gallie et al.,(1989) Molecular Biology of RNA, pages 237-256; and maize chloroticmottle virus leader (MCMV) Lommel et al., Virology 81:382-385 (1991).See also Della-Cioppa et al., Plant Physiology 84:965-968 (1987).

The construct can also contain sequences that enhance translation and/ormRNA stability such as introns. An example of one such intron is thefirst intron of gene II of the histone H3.III variant of Arabidopsisthaliana. Chaubet et al., Journal of Molecular Biology, 225:569-574(1992).

In those instances where it is desirable to have the expressed productof the heterologous nucleotide sequence directed to a particularorganelle, particularly the plastid, amyloplast, or to the endoplasmicreticulum, or secreted at the cell's surface or extracellularly, theexpression cassette can further comprise a coding sequence for a transitpeptide. Such transit peptides are well known in the art and include,but are not limited to, the transit peptide for the acyl carrierprotein, the small subunit of RUBISCO, plant EPSP synthase andHelianthus annuus (U.S. Pat. No. 5,510,417), Zea mays Brittle-1chloroplast transit peptide (Nelson et al., Plant Physiol117(4):1235-1252 (1998); Sullivan et al., Plant Cell 3(12):1337-48;Sullivan et al., Planta (1995) 196(3):477-84; Sullivan et al., J. Biol.Chem. (1992) 267(26):18999-9004) and the like. In addition, chimericchloroplast transit peptides are known in the art, such as the OptimizedTransit Peptide (U.S. Pat. No. 5,510,471). Additional chloroplasttransit peptides have been described previously in U.S. Pat. No.5,717,084 and U.S. Pat. No. 5,728,925. One skilled in the art willreadily appreciate the many options available in expressing a product toa particular organelle. For example, the barley alpha amylase sequenceis often used to direct expression to the endoplasmic reticulum (Rogers,J. Biol. Chem. 260:3731-3738 (1985)).

It will be appreciated by one skilled in the art that use of recombinantDNA technologies can improve control of expression of transfectednucleic acid molecules by manipulating, for example, the number ofcopies of the nucleic acid molecules within the host cell, theefficiency with which those nucleic acid molecules are transcribed, theefficiency with which the resultant transcripts are translated, and theefficiency of post-translational modifications. Additionally, thepromoter sequence might be genetically engineered to improve the levelof expression as compared to the native promoter. Recombinant techniquesuseful for controlling the expression of nucleic acid molecules include,but are not limited to, stable integration of the nucleic acid moleculesinto one or more host cell chromosomes, addition of vector stabilitysequences to plasmids, substitutions or modifications of transcriptioncontrol signals (e.g., promoters, operators, enhancers), substitutionsor modifications of translational control signals (e.g., ribosomebinding sites, Shine-Dalgarno or Kozak sequences), modification ofnucleic acid molecules to correspond to the codon usage of the hostcell, and deletion of sequences that destabilize transcripts.

Reporter or marker genes for selection of transformed cells or tissuesor plant parts or plants can be included in the transformation vectors.Examples of selectable markers include those that confer resistance toanti-metabolites such as herbicides or antibiotics, for example,dihydrofolate reductase, which confers resistance to methotrexate(Reiss, Plant Physiol. (Life Sci. Adv.) 13:143-149, 1994; see alsoHerrera Estrella et al., Nature 303:209-213, (1983); Meijer et al.,Plant Mol. Biol. 16:807-820, (1991)); neomycin phosphotransferase, whichconfers resistance to the aminoglycosides neomycin, kanamycin andparomycin (Herrera-Estrella, EMBO J. 2:987-995, 1983 and Fraley et al.,Proc. Natl. Acad. Sci USA 80:4803 (1983)) and hygromycinphosphotransferase, which confers resistance to hygromycin (Marsh, Gene32:481-485, (1984); see also Waldron et al., Plant Mol. Biol. 5:103-108,(1985); Zhijian et al., Plant Science 108:219-227, (1995)); trpB, whichallows cells to utilize indole in place of tryptophan; hisD, whichallows cells to utilize histinol in place of histidine (Hartman, Proc.Natl. Acad. Sci., USA 85:8047, (1988)); mannose-6-phosphate isomerasewhich allows cells to utilize mannose (International Patent ApplicationNo. WO 94/20627); ornithine decarboxylase, which confers resistance tothe ornithine decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine(DFMO; McConlogue, 1987, In: Current Communications in MolecularBiology, Cold Spring Harbor Laboratory ed.); and deaminase fromAspergillus terreus, which confers resistance to Blasticidin S (Tamura,Biosci. Biotechnol. Biochem. 59:2336-2338, (1995)).

Additional selectable markers include, for example, a mutantacetolactate synthase, which confers imidazolinone or sulfonylurearesistance (Lee et al., EMBO J. 7:1241-1248, (1988)), a mutant psbA,which confers resistance to atrazine (Smeda et al., Plant Physiol.103:911-917, (1993)), or a mutant protoporphyrinogen oxidase (see U.S.Pat. No. 5,767,373), or other markers conferring resistance to anherbicide such as glufosinate. Examples of suitable selectable markergenes include, but are not limited to, genes encoding resistance tochloramphenicol (Herrera Estrella et al., EMBO J. 2:987-992, (1983));streptomycin (Jones et al., Mol. Gen. Genet. 210:86-91, (1987));spectinomycin (Bretagne-Sagnard et al., Transgenic Res. 5:131-137,(1996)); bleomycin (Hille et al., Plant Mol. Biol. 7:171-176, (1990));sulfonamide (Guerineau et al., Plant Mol. Biol. 15:127-136, (1990));bromoxynil (Stalker et al., Science 242:419-423, (1988)); glyphosate(Shaw et al., Science 233:478-481, (1986)); phosphinothricin (DeBlock etal., EMBO J. 6:2513-2518, (1987)), and the like.

One option for use of a selective gene is a glufosinate-resistanceencoding DNA and in one embodiment can be the phosphinothricin acetyltransferase (pat), maize optimized pat gene or bar gene under thecontrol of the Cassava Vein Mosaic Virus promoter. These genes conferresistance to bialaphos. See, (see, Wohlleben et al., (1988) Gene 70:25-37); Gordon-Kamm et al., Plant Cell 2:603; 1990; Uchimiya et al.,BioTechnology 11:835, 1993; White et al., Nucl. Acids Res. 18:1062,1990; Spencer et al., Theor. Appl. Genet. 79:625-631, 1990; and Anzai etal., Mol. Gen. Gen. 219:492, 1989). A version of the pat gene is themaize optimized pat gene, described in U.S. Pat. No. 6,096,947.

In addition, markers that facilitate identification of a plant cellcontaining the polynucleotide encoding the marker may be employed.Scorable or screenable markers are useful, where presence of thesequence produces a measurable product and can produce the productwithout destruction of the plant cell. Examples include aβ-glucuronidase, or uidA gene (GUS), which encodes an enzyme for whichvarious chromogenic substrates are known (for example, U.S. Pat. Nos.5,268,463 and 5,599,670); chloramphenicol acetyl transferase (Jeffersonet al. The EMBO Journal vol. 6 No. 13 pp. 3901-3907); and alkalinephosphatase. In a preferred embodiment, the marker used is beta-caroteneor provitamin A (Ye et al., Science 287:303-305-(2000)). The gene hasbeen used to enhance the nutrition of rice, but in this instance it isemployed instead as a screenable marker, and the presence of the genelinked to a gene of interest is detected by the golden color provided.Unlike the situation where the gene is used for its nutritionalcontribution to the plant, a smaller amount of the protein suffices formarking purposes. Other screenable markers include theanthocyanin/flavonoid genes in general (See discussion at Taylor andBriggs, The Plant Cell (1990)2:115-127) including, for example, aR-locus gene, which encodes a product that regulates the production ofanthocyanin pigments (red color) in plant tissues (Dellaporta et al., inChromosome Structure and Function, Kluwer Academic Publishers, Appelsand Gustafson eds., pp. 263-282 (1988)); the genes which controlbiosynthesis of flavonoid pigments, such as the maize C1 gene (Kao etal., Plant Cell (1996) 8: 1171-1179; Schaller et al., Mol. Gen. Genet.(1994) 242:40-48) and maize C2 (Wienand et al., Mol. Gen. Genet. (1986)203:202-207); the B gene (Chandler et al., Plant Cell (1989)1:1175-1183), the p1 gene (Grotewold et al., Proc. Natl. Acad. Sci USA(1991) 88:4587-4591; Grotewold et al., Cell (1994) 76:543-553; Sidorenkoet al., Plant Mol. Biol. (1999)39:11-19); the bronze locus genes(Ralston et al., Genetics (1988) 119:185-197; Nash et al., Plant Cell(1990) 2(11): 1039-1049), among others.

Further examples of suitable markers include the cyan fluorescentprotein (CYP) gene (Bolte et al., (2004) J. Cell Science 117: 943-54 andKato et al., (2002) Plant Physiol 129: 913-42), the yellow fluorescentprotein gene (PHIYFP™ from Evrogen; see Bolte et al., (2004) J. CellScience 117: 943-54); a lux gene, which encodes a luciferase, thepresence of which may be detected using, for example, X-ray film,scintillation counting, fluorescent spectrophotometry, low-light videocameras, photon counting cameras or multiwell luminometry (Teeri et al.(1989) EMBO J. 8:343); a green fluorescent protein (GFP) gene (Sheen etal., Plant J. (1995) 8(5):777-84); and DsRed2 where plant cellstransformed with the marker gene are red in color, and thus visuallyselectable (Dietrich et al., (2002) Biotechniques 2(2):286-293).Additional examples include a β-lactamase gene (Sutcliffe, Proc. Nat'l.Acad. Sci. U.S.A. (1978) 75:3737), which encodes an enzyme for whichvarious chromogenic substrates are known (e.g., PADAC, a chromogeniccephalosporin); a xylE gene (Zukowsky et al., Proc. Nat'l. Acad. Sci.U.S.A. (1983) 80:1101), which encodes a catechol dioxygenase that canconvert chromogenic catechols; an α-amylase gene (Ikuta et al., Biotech.(1990) 8:241); and a tyrosinase gene (Katz et al., J. Gen. Microbiol.(1983) 129:2703), which encodes an enzyme capable of oxidizing tyrosineto DOPA and dopaquinone, which in turn condenses to form the easilydetectable compound melanin. Clearly, many such markers are availableand known to one skilled in the art.

In certain embodiments, the nucleotide sequence can be optionallycombined with another nucleotide sequence of interest. The term“nucleotide sequence of interest” refers to a nucleic acid molecule(which may also be referred to as a polynucleotide) which can be atranscribed RNA molecule as well as DNA molecule, that encodes for adesired polypeptide or protein, but also may refer to nucleic acidmolecules that do not constitute an entire gene, and which do notnecessarily encode a polypeptide or protein (e.g., a promoter). Forexample, in certain embodiments the nucleic acid molecule can becombined or “stacked” with another that provides additional resistanceor tolerance to glyphosate or another herbicide, and/or providesresistance to select insects or diseases and/or nutritionalenhancements, and/or improved agronomic characteristics, and/or proteinsor other products useful in feed, food, industrial, pharmaceutical orother uses. The “stacking” of two or more nucleic acid sequences ofinterest within a plant genome can be accomplished, for example, viaconventional plant breeding using two or more events, transformation ofa plant with a construct which contains the sequences of interest,re-transformation of a transgenic plant, or addition of new traitsthrough targeted integration via homologous recombination.

Such nucleotide sequences of interest include, but are not limited to,those examples provided below:

1. Genes or Coding Sequence (e.g. iRNA) That Confer Resistance to Pestsor Disease

(A) Plant Disease Resistance Genes. Plant defenses are often activatedby specific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant variety can be transformed with clonedresistance gene to engineer plants that are resistant to specificpathogen strains. Examples of such genes include, the tomato Cf-9 genefor resistance to Cladosporium flavum (Jones et al., 1994 Science266:789), tomato Pto gene, which encodes a protein kinase, forresistance to Pseudomonas syringae pv. tomato (Martin et al., 1993Science 262:1432), and Arabidopsis RSSP2 gene for resistance toPseudomonas syringae (Mindrinos et al., 1994 Cell 78:1089).

(B) A Bacillus thuringiensis protein, a derivative thereof or asynthetic polypeptide modeled thereon, such as, a nucleotide sequence ofa Bt δ-endotoxin gene (Geiser et al., 1986 Gene 48:109), and avegetative insecticidal (VIP) gene (see, e.g., Estruch et al., (1996)Proc. Natl. Acad. Sci. 93:5389-94). Moreover, DNA molecules encodingδ-endotoxin genes can be purchased from American Type Culture Collection(Rockville, Md.), under ATCC accession numbers 40098, 67136, 31995 and31998.

(C) A lectin, such as, nucleotide sequences of several Clivia miniatamannose-binding lectin genes (Van Damme et al., 1994 Plant Molec. Biol.24:825).

(D) A vitamin binding protein, such as avidin and avidin homologs whichare useful as larvicides against insect pests. See U.S. Pat. No.5,659,026.

(E) An enzyme inhibitor, e.g., a protease inhibitor or an amylaseinhibitor. Examples of such genes include a rice cysteine proteinaseinhibitor (Abe et al., 1987 J. Biol. Chem. 262:16793), a tobaccoproteinase inhibitor I (Huub et al., 1993 Plant Molec. Biol. 21:985),and an α-amylase inhibitor (Sumitani et al., 1993 Biosci. Biotech.Biochem. 57:1243).

(F) An insect-specific hormone or pheromone such as an ecdysteroid andjuvenile hormone a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof, such as baculovirus expression of clonedjuvenile hormone esterase, an inactivator of juvenile hormone (Hammocket al., 1990 Nature 344:458).

(G) An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest (J. Biol. Chem. 269:9).Examples of such genes include an insect diuretic hormone receptor(Regan, 1994), an allostatin identified in Diploptera punctata (Pratt,1989), and insect-specific, paralytic neurotoxins (U.S. Pat. No.5,266,361).

(H) An insect-specific venom produced in nature by a snake, a wasp,etc., such as a scorpion insectotoxic peptide (Pang, (1992) Gene116:165).

(I) An enzyme responsible for a hyperaccumulation of monoterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivativeor another non-protein molecule with insecticidal activity.

(J) An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme, anuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. Examples ofsuch genes include, a callas gene (PCT published applicationWO93/02197), chitinase-encoding sequences (which can be obtained, forexample, from the ATCC under accession numbers 3999637 and 67152),tobacco hookworm chitinase (Kramer et al., (1993) Insect Molec. Biol.23:691), and parsley ubi4-2 polyubiquitin gene (Kawalleck et al., (1993)Plant Molec. Biol. 21:673).

(K) A molecule that stimulates signal transduction. Examples of suchmolecules include nucleotide sequences for mung bean calmodulin cDNAclones (Botella et al., (1994) Plant Molec. Biol. 24:757) and anucleotide sequence of a maize calmodulin cDNA clone (Griess et al.,(1994) Plant Physiol. 104:1467).

(L) A hydrophobic moment peptide. See U.S. Pat. Nos. 5,659,026 and5,607,914; the latter teaches synthetic antimicrobial peptides thatconfer disease resistance.

(M) A membrane permease, a channel former or a channel blocker, such asa cecropin-β lytic peptide analog (Jaynes et al., (1993) Plant Sci.89:43) which renders transgenic tobacco plants resistant to Pseudomonassolanacearum.

(N) A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. Coat protein-mediated resistance has beenconferred upon transformed plants against alfalfa mosaic virus, cucumbermosaic virus, tobacco streak virus, potato virus X, potato virus Y,tobacco etch virus, tobacco rattle virus and tobacco mosaic virus. See,for example, Beachy et al., (1990) Ann. Rev. Phytopathol. 28:451.

(O) An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect. Forexample, Taylor et al., (1994) Abstract #497, Seventh Int'l. Symposiumon Molecular Plant-Microbe Interactions shows enzymatic inactivation intransgenic tobacco via production of single-chain antibody fragments.

(P) A virus-specific antibody. See, for example, Tavladoraki et al.,(1993) Nature 266:469, which shows that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

(Q) A developmental-arrestive protein produced in nature by a pathogenor a parasite. Thus, fungal endo α-1,4-D polygalacturonases facilitatefungal colonization and plant nutrient release by solubilizing plantcell wall homo-α-1,4-D-galacturonase (Lamb et al., (1992) Bio/Technology10:1436). The cloning and characterization of a gene which encodes abean endopolygalacturonase-inhibiting protein is described by (Toubartet al., (1992) Plant J. 2:367).

(R) A developmental-arrestive protein produced in nature by a plant,such as the barley ribosome-inactivating gene that provides an increasedresistance to fungal disease (Longemann et al., (1992). Bio/Technology10:3305).

(S) RNA interference, in which a DNA polynucleotide encoding an RNAmolecule is used to inhibit expression of a target gene. An RNA moleculein one example is partially or fully double stranded, which triggers asilencing response, resulting in cleavage of dsRNA into smallinterfering RNAs, which are then incorporated into a targeting complexthat destroys homologous mRNAs. See, e.g., Fire et al., U.S. Pat. No.6,506,559; Graham et al., U.S. Pat. No. 6,573,099.

2. Genes that Confer Resistance to a Herbicide

(A) Genes encoding resistance or tolerance to a herbicide that inhibitsthe growing point or meristem, such as an imidazalinone, sulfonanilideor sulfonylurea herbicide. Exemplary genes in this category code for amutant ALS enzyme (Lee et al., (1988) EMBOJ. 7:1241), which is alsoknown as AHAS enzyme (Miki et al., (1990) Theor. Appl. Genet. 80:449).

(B) One or more additional genes encoding resistance or tolerance toglyphosate imparted by mutant EPSP synthase and aroA genes, or throughmetabolic inactivation by genes such as GAT (glyphosateacetyltransferase) or GOX (glyphosate oxidase) and other phosphonocompounds such as glufosinate (pat and bar genes; DSM-2), andaryloxyphenoxypropionic acids and cyclohexanediones (ACCase inhibitorencoding genes). See, for example, U.S. Pat. No. 4,940,835, whichdiscloses the nucleotide sequence of a form of EPSP which can conferglyphosate resistance. A DNA molecule encoding a mutant aroA gene can beobtained under ATCC Accession Number 39256, and the nucleotide sequenceof the mutant gene is disclosed in U.S. Pat. No. 4,769,061. EuropeanPatent application No. 0 333 033 and U.S. Pat. No. 4,975,374 disclosenucleotide sequences of glutamine synthetase genes which conferresistance to herbicides such as L-phosphinothricin. The nucleotidesequence of a phosphinothricin acetyl-transferase gene is provided inEuropean Patent application No. 0 242 246. De Greef et al., (1989)Bio/Technology 7:61 describes the production of transgenic plants thatexpress chimeric bar genes coding for phosphinothricin acetyltransferase activity. Exemplary of genes conferring resistance toaryloxyphenoxypropionic acids and cyclohexanediones, such as sethoxydimand haloxyfop, are the Accl-S1, Accl-S2 and Accl-S3 genes described byMarshall et al., (1992) Theor. Appl. Genet. 83:435.

(C) Genes encoding resistance or tolerance to a herbicide that inhibitsphotosynthesis, such as a triazine (psbA and gs+ genes) and abenzonitrile (nitrilase gene). Przibilla et al., (1991) Plant Cell 3:169describe the use of plasmids encoding mutant psbA genes to transformChlamydomonas. Nucleotide sequences for nitrilase genes are disclosed inU.S. Pat. No. 4,810,648, and DNA molecules containing these genes areavailable under ATCC accession numbers 53435, 67441 and 67442. Cloningand expression of DNA coding for a glutathione S-transferase isdescribed by Hayes et al., (1992) Biochem. J. 285:173.

(D) Genes encoding resistance or tolerance to a herbicide that bind tohydroxyphenylpyruvate dioxygenases (HPPD), enzymes which catalyze thereaction in which para-hydroxyphenylpyruvate (HPP) is transformed intohomogentisate. This includes herbicides such as isoxazoles (EuropeanPatent No. 418175, European Patent No. 470856, European Patent No.487352, European Patent No. 527036, European Patent No. 560482, EuropeanPatent No. 682659, U.S. Pat. No. 5,424,276), in particular isoxaflutole,which is a selective herbicide for maize, diketonitriles (EuropeanPatent No. 496630, and European Patent No. 496631), in particular2-cyano-3-cyclopropyl-1-(2-SO2CH3-4-CF3 phenyl) propane-1,3-dione and2-cyano-3-cyclopropyl-1-(2-SO2CH3-4-2,3Cl2phenyl) propane-1,3-dione,triketones (European Patent No. 625505, European Patent No. 625508, U.S.Pat. No. 5,506,195), in particular sulcotrione, and pyrazolinates. Agene that produces an overabundance of HPPD in plants can providetolerance or resistance to such herbicides, including, for example,genes described in U.S. Pat. Nos. 6,268,549 and 6,245,968 and U.S.Patent Application, Publication No. 20030066102.

(E) Genes encoding resistance or tolerance to phenoxy auxin herbicides,such as 2,4-dichlorophenoxyacetic acid (2,4-D) and which may also conferresistance or tolerance to aryloxyphenoxypropionate (AOPP) herbicides.Examples of such genes include the α-ketoglutarate-dependent dioxygenaseenzyme (aad-1) gene, described in U.S. Pat. No. 7,838,733.

(F) Genes encoding resistance or tolerance to phenoxy auxin herbicides,such as 2,4-dichlorophenoxyacetic acid (2,4-D) and which may also conferresistance or tolerance to pyridyloxy auxin herbicides, such asfluroxypyr or triclopyr. Examples of such genes include theα-ketoglutarate-dependent dioxygenase enzyme gene (aad-12), described inWO 2007/053482 A2.

(G) Genes encoding resistance or tolerance to dicamba (see, e.g., U.S.Patent Publication No. 20030135879).

(H) Genes providing resistance or tolerance to herbicides that inhibitprotoporphyrinogen oxidase (PPO) (see U.S. Pat. No. 5,767,373).

(I) Genes providing resistance or tolerance to triazine herbicides (suchas atrazine) and urea derivatives (such as diuron) herbicides which bindto core proteins of photosystem II reaction centers (PS II) (SeeBrussian et al., (1989) EMBO J. 1989, 8(4): 1237-1245.

3. Genes That Confer or Contribute to a Value-Added Trait

(A) Modified fatty acid metabolism, for example, by transforming maizeor Brassica with an antisense gene or stearoyl-ACP desaturase toincrease stearic acid content of the plant (Knultzon et al., (1992)Proc. Nat. Acad. Sci. USA 89:2624.

(B) Decreased phytate content

(1) Introduction of a phytase-encoding gene, such as the Aspergillusniger phytase gene (Van Hartingsveldt et al., (1993) Gene 127:87),enhances breakdown of phytate, adding more free phosphate to thetransformed plant.

(2) A gene could be introduced that reduces phytate content. In maize,this, for example, could be accomplished by cloning and thenreintroducing DNA associated with the single allele which is responsiblefor maize mutants characterized by low levels of phytic acid (Raboy etal., (1990) Maydica 35:383).

(C) Modified carbohydrate composition effected, for example, bytransforming plants with a gene coding for an enzyme that alters thebranching pattern of starch. Examples of such enzymes include,Streptococcus mucus fructosyltransferase gene (Shiroza et al., (1988) J.Bacteriol. 170:810), Bacillus subtilis levansucrase gene (Steinmetz etal., (1985) Mol. Gen. Genel. 200:220), Bacillus licheniformis α-amylase(Pen et al., (1992) Bio/Technology 10:292), tomato invertase genes(Elliot et al., (1993), barley amylase gene (Sogaard et al., (1993) J.Biol. Chem. 268:22480), and maize endosperm starch branching enzyme II(Fisher et al., (1993) Plant Physiol. 102:10450).

Described herein are methods of identifying the presence of a donor DNApolynucleotide inserted within a targeted genomic loci. As a furtherembodiment a site specific nuclease can be used to cleave an unmodifiedendogenous plant genomic locus, a previously targeted plant genomiclocus, or a previously inserted exogenous DNA. The targeting of anendogenous genomic loci is one embodiment of the disclosure.

In embodiments, the methods and compositions described herein make useof a site specific nuclease that comprises an engineered (non-naturallyoccurring) Meganuclease (also described as a homing endonuclease). Therecognition sequences of homing endonucleases or meganucleases such asI-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII,I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII and I-TevIII are known. Seealso U.S. Pat. No. 5,420,032; U.S. Pat. No. 6,833,252; Belfort et al.(1997) Nucleic Acids Res. 25:3379-30 3388; Dujon et al. (1989) Gene82:115-118; Perler et al. (1994) Nucleic Acids Res. 22, 11127; Jasin(1996) Trends Genet. 12:224-228; Gimble et al. (1996) J. Mol. Biol.263:163-180; Argast et al. (1998) J. Mol. Biol. 280:345-353 and the NewEngland Biolabs catalogue. In addition, the DNA-binding specificity ofhoming endonucleases and meganucleases can be engineered to bindnon-natural target sites. See, for example, Chevalier et al. (2002)Molec. Cell 10:895-905; Epinat et al. (2003) Nucleic Acids Res. 531:2952-2962; Ashworth et al. (2006) Nature 441:656-659; Paques et al.(2007) Current Gene Therapy 7:49-66; U.S. Patent Publication No.20070117128. The DNA-binding domains of the homing endonucleases andmeganucleases may be altered in the context of the nuclease as a whole(i.e., such that the nuclease includes the cognate cleavage domain) ormay be fused to a heterologous cleavage domain.

In other embodiments, the DNA-binding domain of one or more of thenucleases used in the methods and compositions described hereincomprises a naturally occurring or engineered (non-naturally occurring)TAL effector DNA binding domain. See, e.g., U.S. Patent Publication No.20110301073, incorporated by reference in its entirety herein. The plantpathogenic bacteria of the genus Xanthomonas are known to cause manydiseases in important crop plants. Pathogenicity of Xanthomonas dependson a conserved type III secretion (T3S) system which injects more thandifferent effector proteins into the plant cell. Among these injectedproteins are transcription activator-like (TALEN) effectors which mimicplant transcriptional activators and manipulate the plant transcriptome(see Kay et al (2007) Science 318:648-651). These proteins contain a DNAbinding domain and a transcriptional activation domain. One of the mostwell characterized TAL-effectors is AvrBs3 from Xanthomonas campestgrispv. Vesicatoria (see Bonas et al (1989) Mol Gen Genet 218: 127-136 andWO2010079430). TAL-effectors contain a centralized domain of tandemrepeats, each repeat containing approximately 34 amino acids, which arekey to the DNA binding specificity of these proteins. In addition, theycontain a nuclear localization sequence and an acidic transcriptionalactivation domain (for a review see Schornack S, et al (2006) J PlantPhysiol 163(3): 256-272). In addition, in the phytopathogenic bacteriaRalstonia solanacearum two genes, designated brg11 and hpx17 have beenfound that are homologous to the AvrBs3 family of Xanthomonas in the R.solanacearum biovar strain GMI1000 and in the biovar 4 strain RS1000(See Heuer et al (2007) Appl and Enviro Micro 73(13): 4379-4384). Thesegenes are 98.9% identical in nucleotide sequence to each other butdiffer by a deletion of 1,575 bp in the repeat domain of hpx17. However,both gene products have less than 40% sequence identity with AvrBs3family proteins of Xanthomonas. See, e.g., U.S. Patent Publication No.20110301073, incorporated by reference in its entirety.

Specificity of these TAL effectors depends on the sequences found in thetandem repeats. The repeated sequence comprises approximately 102 bp andthe repeats are typically 91-100% homologous with each other (Bonas etal, ibid). Polymorphism of the repeats is usually located at positions12 and 13 and there appears to be a one-to-one correspondence betweenthe identity of the hypervariable diresidues at positions 12 and 13 withthe identity of the contiguous nucleotides in the TAL-effector's targetsequence (see Moscou and Bogdanove, (2009) Science 326:1501 and Boch etal (2009) Science 326:1509-1512). Experimentally, the natural code forDNA recognition of these TAL-effectors has been determined such that anHD sequence at positions 12 and 13 leads to a binding to cytosine (C),NG binds to T, NI to A, C, G or T, NN binds to A or G, and ING binds toT. These DNA binding repeats have been assembled into proteins with newcombinations and numbers of repeats, to make artificial transcriptionfactors that are able to interact with new sequences and activate theexpression of a non-endogenous reporter gene in plant cells (Boch et al,ibid). Engineered TAL proteins have been linked to a FokI cleavage halfdomain to yield a TAL effector domain nuclease fusion (TALEN) exhibitingactivity in a yeast reporter assay (plasmid based target).

The CRISPR (Clustered Regularly Interspaced Short PalindromicRepeats)/Cas (CRISPR Associated) nuclease system is a recentlyengineered nuclease system based on a bacterial system that can be usedfor genome engineering. It is based on part of the adaptive immuneresponse of many bacteria and Archea. When a virus or plasmid invades abacterium, segments of the invader's DNA are converted into CRISPR RNAs(crRNA) by the ‘immune’ response. This crRNA then associates, through aregion of partial complementarity, with another type of RNA calledtracrRNA to guide the Cas9 nuclease to a region homologous to the crRNAin the target DNA called a “protospacer”. Cas9 cleaves the DNA togenerate blunt ends at the DSB at sites specified by a 20-nucleotideguide sequence contained within the crRNA transcript. Cas9 requires boththe crRNA and the tracrRNA for site specific DNA recognition andcleavage. This system has now been engineered such that the crRNA andtracrRNA can be combined into one molecule (the “single guide RNA”), andthe crRNA equivalent portion of the single guide RNA can be engineeredto guide the Cas9 nuclease to target any desired sequence (see Jinek etal (2012) Science 337, p. 816-821, Jinek et al, (2013), eLife 2:e00471,and David Segal, (2013) eLife 2:e00563). Thus, the CRISPR/Cas system canbe engineered to create a double-stranded break (DSB) at a desiredtarget in a genome, and repair of the DSB can be influenced by the useof repair inhibitors to cause an increase in error prone repair.

In certain embodiments, Cas protein may be a “functional derivative” ofa naturally occurring Cas protein. A “functional derivative” of a nativesequence polypeptide is a compound having a qualitative biologicalproperty in common with a native sequence polypeptide. “Functionalderivatives” include, but are not limited to, fragments of a nativesequence and derivatives of a native sequence polypeptide and itsfragments, provided that they have a biological activity in common witha corresponding native sequence polypeptide. A biological activitycontemplated herein is the ability of the functional derivative tohydrolyze a DNA substrate into fragments. The term “derivative”encompasses both amino acid sequence variants of polypeptide, covalentmodifications, and fusions thereof. Suitable derivatives of a Caspolypeptide or a fragment thereof include but are not limited tomutants, fusions, covalent modifications of Cas protein or a fragmentthereof. Cas protein, which includes Cas protein or a fragment thereof,as well as derivatives of Cas protein or a fragment thereof, may beobtainable from a cell or synthesized chemically or by a combination ofthese two procedures. The cell may be a cell that naturally produces Casprotein, or a cell that naturally produces Cas protein and isgenetically engineered to produce the endogenous Cas protein at a higherexpression level or to produce a Cas protein from an exogenouslyintroduced nucleic acid, which nucleic acid encodes a Cas that is sameor different from the endogenous Cas. In some case, the cell does notnaturally produce Cas protein and is genetically engineered to produce aCas protein. The Cas protein is deployed in mammalian cells (andputatively within plant cells) by co-expressing the Cas nuclease withguide RNA. Two forms of guide RNAs can be ued to facilitate Cas-mediatedgenome cleavage as disclosed in Le Cong, F., et al., (2013) Science339(6121):819-823.

In certain embodiments, the DNA binding domain of one or more of thenucleases used for in vivo cleavage and/or targeted cleavage of thegenome of a cell comprises a zinc finger protein. In some embodiments,the zinc finger protein is non-naturally occurring in that it isengineered to bind to a target site of choice. See, for example, Beerliet al. (2002) Nature Biotechnol. 20:135-141; Pabo et al. (2001) Ann.Rev. Biochem. 70:313-340; Isalan et al. (2001) Nature Biotechnol.19:656-660; Segal et al. (2001) Curr. Opin. Biotechnol. 12:632-637; Chooet al. (2000) Curr. Opin. Struct. Biol. 10:411-416; U.S. Pat. Nos.6,453,242; 6,534,261; 6,599,692; 6,503,717; 6,689,558; 7,030,215;6,794,136; 7,067,317; 7,262,054; 7,070,934; 7,361,635; 7,253,273; andU.S. Patent Publication Nos. 2005/0064474; 2007/0218528; 2005/0267061,all incorporated herein by reference in their entireties.

An engineered zinc finger binding domain can have a novel bindingspecificity, compared to a naturally-occurring zinc finger protein.Engineering methods include, but are not limited to, rational design andvarious types of selection. Rational design includes, for example, usingdatabases comprising triplet (or quadruplet) nucleotide sequences andindividual zinc finger amino acid sequences, in which each triplet orquadruplet nucleotide sequence is associated with one or more amino acidsequences of zinc fingers which bind the particular triplet orquadruplet sequence. See, for example, U.S. Pat. Nos. 6,453,242 and6,534,261, incorporated by reference herein in their entireties.

Selection of target sites; ZFPs and methods for design and constructionof fusion proteins (and polynucleotides encoding same) are known tothose of skill in the art and described in detail in U.S. Pat. Nos.6,140,0815; 789,538; 6,453,242; 6,534,261; 5,925,523; 6,007,988;6,013,453; 6,200,759; WO 95/19431; WO 96/06166; WO 98/53057; WO98/54311; WO 00/27878; WO 01/60970 WO 01/88197; WO 02/099084; WO98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO 03/016496.

In addition, as disclosed in these and other references, zinc fingerdomains and/or multi-fingered zinc finger proteins may be linkedtogether using any suitable linker sequences, including for example,linkers of 5 or more amino acids in length. See, also, U.S. Pat. Nos.6,479,626; 6,903,185; and 7,153,949 for exemplary linker sequences 6 ormore amino acids in length. The proteins described herein may includeany combination of suitable linkers between the individual zinc fingersof the protein.

Thus, the site specific nuclease comprises a DNA-binding domain thatspecifically binds to a target site in any gene into which it is desiredto insert a donor DNA polynucleotide (i.e. comprising at least onetransgene).

Any suitable cleavage domain can be operatively linked to a DNA-bindingdomain to form a nuclease fusion protein. For example, ZFP DNA-bindingdomains have been fused to nuclease domains to create ZFNs—a functionalentity that is able to recognize its intended nucleic acid targetthrough its engineered (ZFP) DNA binding domain and cause the DNA to becut near the ZFP binding site via the nuclease activity. See, e.g., Kimet al. (1996) Proc Natl Acad Sci USA 93(3):1156-1160. More recently,ZFNs have been used for genome modification in a variety of organisms.See, for example, United States Patent Publications 20030232410;20050208489; 20050026157; 20050064474; 20060188987; 20060063231; andInternational Publication WO 07/014275. Likewise, TALEN DNA-bindingdomains have been fused to nuclease domains to create TALENs. See, e.g.,U.S. Publication No. 20110301073.

As noted above, the cleavage domain may be heterologous to theDNA-binding domain, for example a zinc finger DNA-binding domain and acleavage domain from a different nuclease or a TALEN DNA-binding domainand a cleavage domain from a different nuclease, or a meganucleaseDNA-binding domain and cleavage domain from a different nuclease.Heterologous cleavage domains can be obtained from any endonuclease orexonuclease. Exemplary endonucleases from which a cleavage domain can bederived include, but are not limited to, restriction endonucleases andhoming endonucleases. See, for example, 2002-2003 Catalogue, New EnglandBiolabs, Beverly, Mass.; and Belfort et al. (1997) Nucleic Acids Res.25:3379-3388. Additional enzymes which cleave DNA are known (e.g., S1Nuclease; mung bean nuclease; pancreatic DNase I; micrococcal nuclease;yeast HO endonuclease; see also Linn et al. (eds.) Nucleases, ColdSpring Harbor Laboratory Press, 1993). One or more of these enzymes (orfunctional fragments thereof) can be used as a source of cleavagedomains and cleavage half-domains.

Similarly, a cleavage half-domain can be derived from any nuclease orportion thereof, as set forth above, that requires dimerization forcleavage activity. In general, two fusion proteins are required forcleavage if the fusion proteins comprise cleavage half-domains.Alternatively, a single protein comprising two cleavage half-domains canbe used. The two cleavage half-domains can be derived from the sameendonuclease (or functional fragments thereof), or each cleavagehalf-domain can be derived from a different endonuclease (or functionalfragments thereof). In addition, the target sites for the two fusionproteins are preferably disposed, with respect to each other, such thatbinding of the two fusion proteins to their respective target sitesplaces the cleavage half-domains in a spatial orientation to each otherthat allows the cleavage half-domains to form a functional cleavagedomain, e.g., by dimerizing. Thus, in certain embodiments, the nearedges of the target sites are separated by 5-8 nucleotides or by 15-18nucleotides. However any integral number of nucleotides or nucleotidepairs can intervene between two target sites (e.g., from 2 to 50nucleotide pairs or more). In general, the site of cleavage lies betweenthe target sites.

Restriction endonucleases (restriction enzymes) are present in manyspecies and are capable of sequence-specific binding to DNA (at arecognition site), and cleaving DNA at or near the site of binding.Certain restriction enzymes (e.g., Type IIS) cleave DNA at sites removedfrom the recognition site and have separable binding and cleavagedomains. For example, the Type IIS enzyme Fok I catalyzesdouble-stranded cleavage of DNA, at 9 nucleotides from its recognitionsite on one strand and 13 nucleotides from its recognition site on theother. See, for example, U.S. Pat. Nos. 5,356,802; 5,436,150 and5,487,994; as well as Li et al. (1992) Proc. Natl. Acad. Sci. USA89:4275-4279; Li et al. (1993) Proc. Natl. Acad. Sci. USA 90:2764-2768;Kim et al. (1994a) Proc. Natl. Acad. Sci. USA 91:883-887; Kim et al.(1994b) J. Biol. Chem. 269:31,978-31,982. Thus, in one embodiment,fusion proteins comprise the cleavage domain (or cleavage half-domain)from at least one Type IIS restriction enzyme and one or more zincfinger binding domains, which may or may not be engineered.

An exemplary Type IIS restriction enzyme, whose cleavage domain isseparable from the binding domain, is Fok I. This particular enzyme isactive as a dimer. Bitinaite et al., (1998) Proc. Natl. Acad. Sci. USA95: 10,570-10,575. Accordingly, for the purposes of the presentdisclosure, the portion of the Fok I enzyme used in the disclosed fusionproteins is considered a cleavage half-domain. Thus, for targeteddouble-stranded cleavage and/or targeted replacement of cellularsequences using zinc finger-Fok I fusions, two fusion proteins, eachcomprising a FokI cleavage half-domain, can be used to reconstitute acatalytically active cleavage domain. Alternatively, a singlepolypeptide molecule containing a zinc finger binding domain and two FokI cleavage half-domains can also be used. Parameters for targetedcleavage and targeted sequence alteration using zinc finger-Fok Ifusions are provided elsewhere in this disclosure.

A cleavage domain or cleavage half-domain can be any portion of aprotein that retains cleavage activity, or that retains the ability tomultimerize (e.g., dimerize) to form a functional cleavage domain.

Exemplary Type IIS restriction enzymes are described in InternationalPatent Application Publication WO 07/014275, incorporated herein in itsentirety. Additional restriction enzymes also contain separable bindingand cleavage domains, and these are contemplated by the presentdisclosure. See, for example, Roberts et al. (2003) Nucleic Acids Res.31:418-420.

In certain embodiments, the cleavage domain comprises one or moreengineered cleavage half-domain (also referred to as dimerization domainmutants) that minimize or prevent homodimerization, as described, forexample, in U.S. Patent Publication Nos. 20050064474; 20060188987;20070305346 and 20080131962, the disclosures of all of which areincorporated by reference in their entireties herein. Amino acidresidues at positions 446, 447, 479, 483, 484, 486, 487, 490, 491, 496,498, 499, 500, 531, 534, 537, and 538 of Fok I are all targets forinfluencing dimerization of the Fok I cleavage half-domains.

Exemplary engineered cleavage half-domains of Fok I that form obligateheterodimers include a pair in which a first cleavage half-domainincludes mutations at amino acid residues at positions 490 and 538 ofFok I and a second cleavage half-domain includes mutations at amino acidresidues 486 and 499.

Thus, in one embodiment, a mutation at 490 replaces Glu (E) with Lys(K); the mutation at 538 replaces Iso (I) with Lys (K); the mutation at486 replaced Gln (Q) with Glu (E); and the mutation at position 499replaces Iso (I) with Lys (K). Specifically, the engineered cleavagehalf-domains described herein were prepared by mutating positions 490(E→K) and 538 (I→K) in one cleavage half-domain to produce an engineeredcleavage half-domain designated “E490K:I538K” and by mutating positions486 (Q→E) and 499 (I→L) in another cleavage half-domain to produce anengineered cleavage half-domain designated “Q486E:1499L”. The engineeredcleavage half-domains described herein are obligate heterodimer mutantsin which aberrant cleavage is minimized or abolished. See, e.g., U.S.Patent Publication No. 2008/0131962, the disclosure of which isincorporated by reference in its entirety for all purposes. In certainembodiments, the engineered cleavage half-domain comprises mutations atpositions 486, 499 and 496 (numbered relative to wild-type FokI), forinstance mutations that replace the wild type Gln (Q) residue atposition 486 with a Glu (E) residue, the wild type Iso (I) residue atposition 499 with a Leu (L) residue and the wild-type Asn (N) residue atposition 496 with an Asp (D) or Glu (E) residue (also referred to as a“ELD” and “ELE” domains, respectively). In other embodiments, theengineered cleavage half-domain comprises mutations at positions 490,538 and 537 (numbered relative to wild-type FokI), for instancemutations that replace the wild type Glu (E) residue at position 490with a Lys (K) residue, the wild type Iso (I) residue at position 538with a Lys (K) residue, and the wild-type His (H) residue at position537 with a Lys (K) residue or a Arg (R) residue (also referred to as“KKK” and “KKR” domains, respectively). In other embodiments, theengineered cleavage half-domain comprises mutations at positions 490 and537 (numbered relative to wild-type FokI), for instance mutations thatreplace the wild type Glu (E) residue at position 490 with a Lys (K)residue and the wild-type His (H) residue at position 537 with a Lys (K)residue or a Arg (R) residue (also referred to as “KIK” and “KIR”domains, respectively). (See US Patent Publication No. 20110201055). Inother embodiments, the engineered cleavage half domain comprises the“Sharkey” and/or “Sharkey” mutations (see Guo et al, (2010) J. Mol.Biol. 400(1):96-107).

Engineered cleavage half-domains described herein can be prepared usingany suitable method, for example, by site-directed mutagenesis ofwild-type cleavage half-domains (Fok I) as described in U.S. PatentPublication Nos. 20050064474; 20080131962; and 20110201055.

Alternatively, nucleases may be assembled in vivo at the nucleic acidtarget site using so-called “split-enzyme” technology (see e.g. U.S.Patent Publication No. 20090068164). Components of such split enzymesmay be expressed either on separate expression constructs, or can belinked in one open reading frame where the individual components areseparated, for example, by a self-cleaving 2A peptide or IRES sequence.Components may be individual zinc finger binding domains or domains of ameganuclease nucleic acid binding domain.

Nucleases can be screened for activity prior to use, for example in ayeast-based chromosomal system as described in WO 2009/042163 and20090068164. Nuclease expression constructs can be readily designedusing methods known in the art. See, e.g., United States PatentPublications 20030232410; 20050208489; 20050026157; 20050064474;20060188987; 20060063231; and International Publication WO 07/014275.Expression of the nuclease may be under the control of a constitutivepromoter or an inducible promoter.

A “target” or “target site” or “targeted genomic locus” is a nucleicacid sequence that defines a portion of a nucleic acid to which abinding molecule (e.g. site specific nuclease) will bind, providedsufficient conditions for binding exist.

In an embodiment a genomic locus sequence includes those present inchromosomes, episomes, organellar genomes (e.g., mitochondria,chloroplasts), artificial chromosomes and any other type of nucleic acidpresent in a cell such as, for example, amplified sequences, doubleminute chromosomes and the genomes of endogenous or infecting bacteriaand viruses. Genomic locus sequences can be normal (i.e., wild-type) ormutant; mutant sequences can comprise, for example, insertions (e.g.,previously inserted exogenous polynucleotides), deletions,translocations, rearrangements, and/or point mutations. A genomic locussequence can also comprise one of a number of different alleles.

Also described herein as an embodiment of the invention are methods forinserting a donor DNA polynucleotide sequence within a genomic loci.Reported and observed frequencies of targeted genomic modificationindicate that targeting of a genomic loci within plants is relativelyinefficient. The success rate of such methods are low, due in part topoor efficiency of homologous recombination and a high frequency ofnon-specific insertion of the donor DNA into regions of the genome otherthan the target site. The present disclosure provides methods foridentifying a donor DNA polynucleotide within a targeted genomic loci

The methods of the subject disclosure involve making and using sitespecific nucleases (e.g., engineered zinc finger binding domains fusedto cleavage domains) to make one or more targeted double-stranded breaksin cellular DNA. Because double-stranded breaks in cellular DNAstimulate cellular repair mechanisms several thousand-fold in thevicinity of the cleavage site, such targeted cleavage allows for thealteration or replacement (via homology-directed repair) of sequences atvirtually any site in the genome.

In addition to the fusion molecules described herein, targetedreplacement of a selected genomic sequence also requires theintroduction of the replacement or donor DNA polynucleotide sequence.The donor DNA polynucleotide sequence can be introduced into the cellprior to, concurrently with, or subsequent to, expression of the fusionprotein(s). The donor DNA polynucleotide contains sufficient homology toa genomic sequence to support homologous recombination (orhomology-directed repair) between it and the genomic sequence to whichit bears homology. Approximately 25, 50 100, 200, 500, 750, 1,000,1,500, 2,000 nucleotides or more of sequence homology between a donorDNA polynucleotide and a genomic locus (or any integral value between 10and 2,000 nucleotides, or more) will support homologous recombinationtherebetween. Donor DNA polynucleotide sequences can range in lengthfrom 10 to 5,000 nucleotides (or any integral value of nucleotidestherebetween) or longer. It will be readily apparent that the donor DNApolynucleotide sequence is typically not identical to the genomicsequence that it replaces. For example, the sequence of the donor DNApolynucleotide can contain one or more single base changes, insertions,deletions, inversions or rearrangements with respect to the genomicsequence, so long as sufficient homology with chromosomal sequences ispresent. Alternatively, a donor DNA polynucleotide sequence can containa non-homologous sequence flanked by two regions of homology.Additionally, donor DNA polynucleotide sequences can comprise a vectormolecule containing sequences that are not homologous to the region ofinterest in cellular chromatin. Generally, the homologous region(s) of adonor DNA polynucleotide sequence will have at least 50% sequenceidentity to a genomic locus with which recombination is desired. Incertain embodiments, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 99.9%sequence identity is present. Any value between 1% and 100% sequenceidentity can be present, of depending upon the length the donor DNApolynucleotide.

A donor DNA polynucleotide molecule can contain several, discontinuousregions of homology to cellular chromatin. For example, for targetedinsertion of sequences not normally present in a region of interest,said sequences can be present in a donor DN polynucleic acid moleculeand flanked by regions of homology to sequence in the region ofinterest.

The donor polynucleotide can be DNA or RNA, single-stranded ordouble-stranded and can be introduced into a cell in linear or circularform. If introduced in linear form, the ends of the donor sequence canbe protected (e.g., from exonucleolytic degradation) by methods known tothose of skill in the art. For example, one or more dideoxynucleotideresidues are added to the 3′ terminus of a linear molecule and/orself-complementary oligonucleotides are ligated to one or both ends.See, for example, Chang et al., (1987) Proc. Natl Acad. Sd. USA84:4959-4963; Nehls et al., (1996) Science 272:886-889. Additionalmethods for protecting exogenous polynucleotides from degradationinclude, but are not limited to, addition of terminal amino group(s) andthe use of modified internucleotide linkages such as, for example,phosphorothioates, phosphoramidates, and O-methyl ribose or deoxyriboseresidues.

A donor DNA polynucleotide can be introduced into a cell as part of avector molecule having additional sequences such as, for example,replication origins, promoters and genes encoding antibiotic resistance.Moreover, donor DNA polynucleotides can be introduced as naked nucleicacid, as nucleic acid complexed with an agent such as a liposome orpoloxamer or can be delivered by bacteria or viruses (e.g.,Agrobacterium sp., Rhizobium sp. NGR234, Sinorhizoboium meliloti,Mesorhizobium loti, tobacco mosaic virus, potato virus X, cauliflowermosaic virus and cassava vein mosaic virus. See, e.g., Chung et al.(2006) Trends Plant Sd. 11(1): 1-4)

Without being bound by one theory, it appears that the presence of adouble-stranded break in a cellular sequence, coupled with the presenceof an exogenous DNA molecule having homology to a region adjacent to orsurrounding the break, activates cellular mechanisms which repair thebreak by transfer of sequence information from the donor molecule intothe cellular {e.g., genomic or chromosomal) sequence; i.e., by aprocesses of homology-directed repair, also known as “gene conversion.”Applicants' methods advantageously combine the powerful targetingcapabilities of engineered ZFPs with a cleavage domain (or cleavagehalf-domain) to specifically target a double-stranded break to theregion of the genome at insertion of exogenous sequences is desired.

For alteration of a chromosomal sequence, it is not necessary for theentire sequence of the donor to be copied into the chromosome, as longas enough of the donor sequence is copied to effect the desired sequencealteration.

The efficiency of insertion of donor sequences by homologousrecombination is inversely related to the distance, in the cellular DNA,between the double-stranded break and the site at which recombination isdesired. In other words, higher homologous recombination efficienciesare observed when the double-stranded break is closer to the site atwhich recombination is desired. In cases in which a precise site ofrecombination is not predetermined (e.g., the desired recombinationevent can occur over an interval of genomic sequence), the length andsequence of the donor nucleic acid, together with the site(s) ofcleavage, are selected to obtain the desired recombination event. Incases in which the desired event is designed to change the sequence of asingle nucleotide pair in a genomic sequence, cellular chromatin iscleaved within 10,000 nucleotides on either side of that nucleotidepair. In certain embodiments, cleavage occurs within 1,000, 500, 200,100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, or 2 nucleotides, or anyintegral value between 2 and 1,000 nucleotides, on either side of thenucleotide pair whose sequence is to be changed.

As detailed above, the binding sites for two fusion proteins, eachcomprising a zinc finger binding domain and a cleavage half-domain, canbe located 5-8 or 15-18 nucleotides apart, as measured from the edge ofeach binding site nearest the other binding site, and cleavage occursbetween the binding sites. Whether cleavage occurs at a single site orat multiple sites between the binding sites is immaterial, since thecleaved genomic sequences are replaced by the donor sequences. Thus, forefficient alteration of the sequence of a single nucleotide pair bytargeted recombination, the midpoint of the region between the bindingsites is within 10,000 nucleotides of that nucleotide pair, preferablywithin 1,000 nucleotides, or 500 nucleotides, or 200 nucleotides, or 100nucleotides, or 50 nucleotides, or 20 nucleotides, or 10 nucleotides, or5 nucleotide, or 2 nucleotides, or one nucleotide, or at the nucleotidepair of interest.

In certain embodiments, a homologous chromosome can serve as the donorDNA polynucleotide. Thus, for example, correction of a mutation in aheterozygote can be achieved by engineering fusion proteins which bindto and cleave the mutant sequence on one chromosome, but do not cleavethe wild-type sequence on the homologous chromosome. The double-strandedbreak on the mutation-bearing chromosome stimulates a homology-based“gene conversion” process in which the wild-type sequence from thehomologous chromosome is copied into the cleaved chromosome, thusrestoring two copies of the wild-type sequence.

Methods and compositions are also provided that may enhance levels oftargeted recombination including, but not limited to, the use ofadditional ZFP-functional domain fusions to activate expression of genesinvolved in homologous recombination, such as, for example, members ofthe RAD52 epistasis group (e.g., Rad50, Rad51, Rad51B, RadSIC, RadSID,Rad52, Rad54, Rad54B, Mrell, XRCC2, XRCC3), genes whose productsinteract with the aforementioned gene products (e.g., BRCA1, BRCA2)and/or genes in the NBS1 complex. See, e.g., Boyko et al. (2006) PlantPhysiology 141:488-497 and LaFarge et al. (2003) Nucleic Acids Res31(4): 1148-1155. Similarly ZFP-functional domain fusions can be used,in combination with the methods and compositions disclosed herein, torepress expression of genes involved in non-homologous end joining(e.g., Ku70/80, XRCC4, poly(ADP ribose) polymerase, DNA ligase 4). See,for example, Riha et al. (2002) EMBO 21:2819-2826; Freisner et al.(2003) Plant J. 34:427-440; Chen et al. (1994) European Journal ofBiochemistry 224:135-142. Methods for activation and repression of geneexpression using fusions between a zinc finger binding domain and afunctional domain are disclosed, for example, in co-owned U.S. Pat. Nos.6,534,261; 6,824,978 and 6,933,113. Additional repression methodsinclude the use of antisense oligonucleotides and/or small interferingRNA (siRNA or RNAi) targeted to the sequence of the gene to berepressed.

The genetic manipulations of a recombinant host disclosed herein can beperformed using standard genetic techniques in any host cell that issuitable to genetic manipulation. In some embodiments, a recombinanthost cell disclosed herein can be any organism or microorganism hostuseful for genetic modification and recombinant gene expression. In someembodiments, a recombinant host can be but is not limited to any higherplant, including both dicotyledonous and monocotyledonous plants, andconsumable plants, including crop plants and plants used for their oils.Thus, any plant species or plant cell can be selected as describedfurther below.

In some embodiments, plants which comprise a donor DNA polynucleotideinserted within a targeted genomic locus in accordance with the presentdisclosure (e.g., plant host cells) include, but is not limited to, anyhigher plants, including both dicotyledonous and monocotyledonousplants, and particularly consumable plants, including crop plants. Suchplants can include, but are not limited to, for example: alfalfa,soybeans, cotton, rapeseed (also described as canola), linseed, corn,rice, brachiaria, wheat, safflowers, sorghum, sugarbeet, sunflowers,tobacco and turf grasses. Thus, any plant species or plant cell can beselected. In embodiments, plant cells used herein, and plants grown orderived therefrom, include, but are not limited to, cells obtainablefrom rapeseed (Brassica napus); indian mustard (Brassica juncea);Ethiopian mustard (Brassica carinata); turnip (Brassica rapa); cabbage(Brassica oleracea); soybean (Glycine max); linseed/flax (Linumusitatissimum); maize (also described as corn) (Zea mays); safflower(Carthamus tinctorius); sunflower (Helianthus annuus); tobacco(Nicotiana tabacum); Arabidopsis thaliana; Brazil nut (Betholettiaexcelsa); castor bean (Ricinus communis); coconut (Cocus nucifera);coriander (Coriandrum sativum); cotton (Gossypium spp.); groundnut(Arachis hypogaea); jojoba (Simmondsia chinensis); oil palm (Elaeisguineeis); olive (Olea eurpaea); rice (Oryza sativa); squash (Cucurbitamaxima); barley (Hordeum vulgare); sugarcane (Saccharum officinarum);rice (Oryza sativa); wheat (Triticum spp. including Triticum durum andTriticum aestivum); and duckweed (Lemnaceae sp.). In some embodiments,the genetic background within a plant species may vary.

“Plant parts,” as used herein, include any parts of a plant, including,but not limited to, seeds (including mature seeds and immature seeds), aplant cutting, a plant cell, a plant cell culture, a plant organ,pollen, embryos, flowers, fruits, shoots, leaves, roots, stems,explants, etc. A plant cell is the structural and physiological unit ofthe plant, comprising a protoplast and a cell wall. A plant cell can bein the form of an isolated single cell or aggregate of cells such as afriable callus, or a cultured cell, or can be part of a higher organizedunit, for example, a plant tissue, plant organ, or plant. Thus, a plantcell can be a protoplast, a gamete producing cell, or a cell orcollection of cells that can regenerate into a whole plant. As such, aseed, which comprises multiple plant cells and is capable ofregenerating into a whole plant, is considered a plant cell for purposesof this disclosure. A plant tissue or plant organ can be a seed,protoplast, callus, or any other groups of plant cells that is organizedinto a structural or functional unit. Particularly useful parts of aplant include harvestable parts and parts useful for propagation ofprogeny plants. A harvestable part of a plant can be any useful part ofa plant, for example, flowers, pollen, seedlings, tubers, leaves, stems,fruit, seeds, roots, and the like. A part of a plant useful forpropagation includes, for example, seeds, fruits, cuttings, seedlings,tubers, rootstocks, and the like. The tissue culture will preferably becapable of regenerating plants having the physiological andmorphological characteristics of the foregoing inbred plant, and ofregenerating plants having substantially the same genotype as theforegoing inbred plant. In an embodiment, the regenerable cells in suchtissue cultures will be embryos, protoplasts, meristematic cells,callus, pollen, leaves, anthers, roots, root tips, silk, flowers,kernels, ears, cobs, husks or stalks. Still further, embodiments of thepresent disclosure provide plants regenerated from the tissue culturesof embodiments of the disclosure.

With regard to the production of plants comprising a donor DNApolynucleotide inserted within a genomic locus, methods for thetransformation of plants are well known in the art. For instance,numerous methods for plant transformation have been developed, includingbiological and physical transformation protocols for dicotyledenousplants as well as monocotyledenous plants (e.g., Goto-Fumiyuki et al.,Nature Biotech 17:282-286 (1999); Miki et al., Methods in PlantMolecular Biology and Biotechnology, Glick, B. R. and Thompson, J. E.Eds., CRC Press, Inc., Boca Raton, pp. 67-88 (1993)). In addition,vectors comprising gene expression cassettes and in vitro culturemethods for plant cell or tissue transformation and regeneration ofplants are available, for example, in Gruber et al., Methods in PlantMolecular Biology and Biotechnology, Glick, B. R. and Thompson, J. E.Eds., CRC Press, Inc., Boca Raton, pp. 89-119 (1993).

A large number of techniques are available for inserting DNA comprisinga gene expression cassette into a plant host cell. Those techniquesinclude transformation with disarmed T-DNA using Agrobacteriumtumefaciens or Agrobacterium rhizogenes as the transformation agent,calcium phosphate transfection, polybrene transformation, protoplastfusion, electroporation, ultrasonic methods (e.g., sonoporation),liposome transformation, microinjection, naked DNA, plasmid vectors,viral vectors, biolistics (microparticle bombardment), silicon carbideWHISKERS™ mediated transformation, aerosol beaming, or Poly EthyleneGlycol mediated transformation as well as other possible methods.

For example, the DNA construct comprising a gene expression cassette maybe introduced directly into the genomic DNA of the plant cell usingtechniques such as electroporation and microinjection of plant cellprotoplasts, or the DNA constructs can be introduced directly to planttissue using biolistic methods, such as DNA particle bombardment (see,e.g., Klein et al. (1987) Nature 327:70-73). Additional methods forplant cell transformation include microinjection via silicon carbideWHISKERS™ mediated DNA uptake (Kaeppler et al. (1990) Plant CellReporter 9:415-418). Alternatively, the DNA construct can be introducedinto the plant cell via nanoparticle transformation (see, e.g., U.S.patent application Ser. No. 12/245,685, which is incorporated herein byreference in its entirety).

Another known method of plant transformation is microprojectile-mediatedtransformation wherein DNA is carried on the surface ofmicroprojectiles. In this method, the expression vector is introducedinto plant tissues with a biolistic device that accelerates themicroprojectiles to speeds sufficient to penetrate plant cell walls andmembranes. Sanford et al., Part. Sci. Technol. 5:27 (1987), Sanford, J.C., Trends Biotech. 6:299 (1988), Sanford, J. C., Physiol. Plant 79:206(1990), Klein et al., Biotechnology 10:268 (1992).

Alternatively, gene transfer and transformation methods include, but arenot limited to, protoplast transformation through calcium chlorideprecipitation, poly ethylene glycol (PEG) or electroporation-mediateduptake of DNA (see Paszkowski et al. (1984) EMBO J 3:2717-2722, Potrykuset al. (1985) Molec. Gen. Genet. 199:169-177; Fromm et al. (1985) Proc.Nat. Acad. Sci. USA 82:5824-5828; and Shimamoto (1989) Nature338:274-276) and electroporation of plant tissues (D'Halluin et al.(1992) Plant Cell 4:1495-1505).

A widely utilized method for introducing an vector comprising a geneexpression cassette into plants is based on the natural transformationsystem of Agrobacterium. Horsch et al., Science 227:1229 (1985). A.tumefaciens and A. rhizogenes are plant pathogenic soil bacteria knownto be useful to genetically transform plant cells. The Ti and Riplasmids of A. tumefaciens and A. rhizogenes, respectively, carry genesresponsible for genetic transformation of the plant. Kado, C. I., Crit.Rev. Plant. Sci. 10:1 (1991). Descriptions of Agrobacterium vectorsystems and methods for Agrobacterium-mediated gene transfer are alsoavailable, for example, Gruber et al., supra, Miki et al., supra,Moloney et al., Plant Cell Reports 8:238 (1989), and U.S. Pat. Nos.4,940,838 and 5,464,763.

If Agrobacterium is used for the transformation, the DNA to be insertedshould be cloned into special plasmids, namely either into anintermediate vector or into a binary vector. Intermediate vectors cannotreplicate themselves in Agrobacterium. The intermediate vector can betransferred into Agrobacterium tumefaciens by means of a helper plasmid(conjugation). The Japan Tobacco Superbinary system is an example ofsuch a system (reviewed by Komari et al., (2006) In: Methods inMolecular Biology (K. Wang, ed.) No. 343: Agrobacterium Protocols(2^(nd) Edition, Vol. 1) HUMANA PRESS Inc., Totowa, N.J., pp. 15-41; andKomori et al., (2007) Plant Physiol. 145:1155-1160). Binary vectors canreplicate in both E. coli and in Agrobacterium. They comprise aselection marker gene and a linker or polylinker which are framed by theright and left T-DNA border regions. They can be transformed directlyinto Agrobacterium (Holsters, 1978). The Agrobacterium used as host cellis to comprise a plasmid carrying a vir region. The Ti or Ri plasmidalso comprises the vir region necessary for the transfer of the T-DNA.The vir region is necessary for the transfer of the T-DNA into the plantcell. Additional T-DNA may be contained.

The virulence functions of the Agrobacterium tumefaciens host willdirect the insertion of a T-strand containing the construct and adjacentmarker into the plant cell DNA when the cell is infected by the bacteriausing a binary T DNA vector (Bevan (1984) Nuc. Acid Res. 12:8711-8721)or the co-cultivation procedure (Horsch et al. (1985) Science227:1229-1231). Generally, the Agrobacterium transformation system isused to engineer dicotyledonous plants (Bevan et al. (1982) Ann. Rev.Genet 16:357-384; Rogers et al. (1986) Methods Enzymol. 118:627-641).The Agrobacterium transformation system may also be used to transform,as well as transfer, DNA to monocotyledonous plants and plant cells. SeeU.S. Pat. No. 5,591,616; Hernalsteen et al. (1984) EMBO J 3:3039-3041;Hooykass-Van Slogteren et al. (1984) Nature 311:763-764; Grimsley et al.(1987) Nature 325:1677-179; Boulton et al. (1989) Plant Mol. Biol.12:31-40; and Gould et al. (1991) Plant Physiol. 95:426-434.

Following the introduction of the genetic construct comprising a geneexpression cassette into plant cells, plant cells can be grown and uponemergence of differentiating tissue such as shoots and roots, matureplants can be generated. In some embodiments, a plurality of plants canbe generated. Methodologies for regenerating plants are known to thoseof ordinary skill in the art and can be found, for example, in: PlantCell and Tissue Culture, 1994, Vasil and Thorpe Eds. Kluwer AcademicPublishers and in: Plant Cell Culture Protocols (Methods in MolecularBiology 111, 1999 Hall Eds Humana Press). The genetically modified plantdescribed herein can be cultured in a fermentation medium or grown in asuitable medium such as soil. In some embodiments, a suitable growthmedium for higher plants can include any growth medium for plants,including, but not limited to, soil, sand, any other particulate mediathat support root growth (e.g., vermiculite, perlite, etc.) orhydroponic culture, as well as suitable light, water and nutritionalsupplements which optimize the growth of the higher plant.

Transformed plant cells which are produced by any of the abovetransformation techniques can be cultured to regenerate a whole plantwhich possesses the transformed genotype and thus the desired phenotype.Such regeneration techniques rely on manipulation of certainphytohormones in a tissue culture growth medium, typically relying on abiocide and/or herbicide marker which has been introduced together withthe desired nucleotide sequences. Plant regeneration from culturedprotoplasts is described in Evans, et al., “Protoplasts Isolation andCulture” in Handbook of Plant Cell Culture, pp. 124-176, MacmillianPublishing Company, New York, 1983; and Binding, Regeneration of Plants,Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. Regenerationcan also be obtained from plant callus, explants, organs, pollens,embryos or parts thereof. Such regeneration techniques are describedgenerally in Klee et al. (1987) Ann. Rev. of Plant Phys. 38:467-486.

Nucleic acids introduced into a plant cell can be used to confer desiredtraits on essentially any plant. A wide variety of plants and plant cellsystems may be engineered for the desired physiological and agronomiccharacteristics described herein using the nucleic acid constructs ofthe present disclosure and the various transformation methods mentionedabove. In preferred embodiments, plants and plant cells for engineeringinclude, but are not limited to, those monocotyledonous anddicotyledonous plants, such as crops including grain crops (e.g., wheat,maize, rice, millet, barley), fruit crops (e.g., tomato, apple, pear,strawberry, orange), forage crops (e.g., alfalfa), root vegetable crops(e.g., carrot, potato, sugar beets, yam), leafy vegetable crops (e.g.,lettuce, spinach); flowering plants (e.g., petunia, rose,chrysanthemum), conifers and pine trees (e.g., pine fir, spruce); plantsused in phytoremediation (e.g., heavy metal accumulating plants); oilcrops (e.g., sunflower, rape seed) and plants used for experimentalpurposes (e.g., Arabidopsis). Thus, the disclosed methods andcompositions have use over a broad range of plants, including, but notlimited to, species from the genera Asparagus, Avena, Brassica, Citrus,Citrullus, Capsicum, Cucurbita, Daucus, Erigeron, Glycine, Gossypium,Hordeum, Lactuca, Lolium, Lycopersicon, Malus, Manihot, Nicotiana,Orychophragmus, Oryza, Persea, Phaseolus, Pisum, Pyrus, Prunus,Raphanus, Secale, Solanum, Sorghum, Triticum, Vitis, Vigna, and Zeamays.

A transformed plant cell, callus, tissue or plant may be identified andisolated by selecting or screening the engineered plant material fortraits encoded by the marker genes present on the transforming DNA. Forinstance, selection can be performed by growing the engineered plantmaterial on media containing an inhibitory amount of the antibiotic orherbicide to which the transforming gene construct confers resistance.Further, transformed plants and plant cells can also be identified byscreening for the activities of any visible marker genes (e.g., theβ-glucuronidase, luciferase, or gfp genes) that may be present on therecombinant nucleic acid constructs. Such selection and screeningmethodologies are well known to those skilled in the art.

The term “introduced” in the context of inserting a nucleic acid into acell, includes transformation into the cell, as well as crossing a planthaving the sequence with another plant, so that the second plantcontains the heterologous sequence, as in conventional plant breedingtechniques. Such breeding techniques are well known to one skilled inthe art. For a discussion of plant breeding techniques, see Poehlman(1995) Breeding Field Crops. AVI Publication Co., Westport Conn., 4^(th)Edit. Backcrossing methods may be used to introduce a gene into theplants. This technique has been used for decades to introduce traitsinto a plant. An example of a description of this and other plantbreeding methodologies that are well known can be found in referencessuch as Poehlman, supra, and Plant Breeding Methodology, edit. NealJensen, John Wiley & Sons, Inc. (1988). In a typical backcross protocol,the original variety of interest (recurrent parent) is crossed to asecond variety (nonrecurrent parent) that carries the single gene ofinterest to be transferred. The resulting progeny from this cross arethen crossed again to the recurrent parent and the process is repeateduntil a plant is obtained wherein essentially all of the desiredmorphological and physiological characteristics of the recurrent parentare recovered in the converted plant, in addition to the singletransferred gene from the nonrecurrent parent.

The term transgenic “event” refers to a recombinant plant produced bytransformation and regeneration of a single plant cell with heterologousDNA, for example, an expression cassette that includes a gene ofinterest. The term “event” refers to the original transformant and/orprogeny of the transformant that include the heterologous DNA. The term“event” also refers to progeny produced by a sexual outcross between thetransformant and another plant. Even after repeated backcrossing to arecurrent parent, the inserted DNA and the flanking DNA from thetransformed parent is present in the progeny of the cross at the samechromosomal location. Normally, transformation of plant tissue producesmultiple events, each of which represent insertion of a DNA constructinto a different location in the genome of a plant cell. Based on theexpression of the transgene or other desirable characteristics, aparticular event is selected. In embodiments of the subject disclosurethe particular event comprises a donor DNA polynucleotide insertedwithin a targeted genomic locus.

A “transgene” refers to a gene introduced into the genome of an organismby genetic manipulation in order to alter its genotype.

A “transgenic plant” is a plant having one or more plant cells thatcontain an expression vector comprising a gene expression cassette. Theterm “Messenger RNA (mRNA)” refers to the RNA that is without intronsand that can be translated into protein by the cell.

As used herein, “insert DNA” refers to the heterologous DNA within thedonor DNA polynucleotide comprising a gene expression cassette used totransform the plant material while “flanking DNA” or “junction DNA” cancomprise either genomic DNA naturally present in an organism such as aplant, or foreign (heterologous) DNA introduced via the transformationprocess which is extraneous to the original insert DNA molecule, e.g.fragments associated with the transformation event. A “junction” or“flanking region” or “flanking sequence” as used herein refers to asequence of at least 20, 50, 100, 200, 300, 400, 1000, 1500, 2000, 2500,or 5000 base pair or greater which is located either immediatelyupstream of and contiguous with or immediately downstream of andcontiguous with the original foreign insert DNA molecule.

In an embodiment the disclosure relates to a method to identify thepresence of a donor DNA polynucleotide within a targeted genome via anamplification reaction in which an amplicon is generated. The detectionof the absence of the amplicon is an indication of whether the genomicloci has been disrupted. In additional embodiments, the presence of anamplicon is an indication that a donor DNA polynucleotide was insertedwithin the genomic loci.

Various assays can be employed in connection with the amplificationreaction of certain embodiments of the disclosure. The followingtechniques are useful in a variety of situations, and in one embodiment,are useful in detecting the presence of the nucleic acid molecule and/orthe polypeptide encoded in a plant cell. For example, the presence ofthe molecule can be determined in a variety of ways, including using aprimer or probe of the sequence. The transgene may be selectivelyexpressed in some tissues of the plant or at some developmental stages,or the transgene may be expressed in substantially all plant tissues,substantially along its entire life cycle. However, any combinatorialexpression mode is also applicable.

Amplification of a selected, or target, nucleic acid sequence may becarried out by any suitable means. See generally, Kwoh et al., Am.Biotechnol. Lab. 8, 14-25 (1990). Examples of suitable amplificationtechniques include, but are not limited to, polymerase chain reaction,ligase chain reaction, strand displacement amplification (see generallyG. Walker et al., Proc. Natl. Acad. Sci. USA 89, 392-396 (1992); G.Walker et al., Nucleic Acids Res. 20, 1691-1696 (1992)),transcription-based amplification (see D. Kwoh et al., Proc. Natl. AcadSci. USA 86, 1173-1177 (1989)), self-sustained sequence replication (or“35R”) (see J. Guatelli et al., Proc. Natl. Acad. Sci. USA 87, 1874-1878(1990)), the Qβ replicase system (see P. Lizardi et al., BioTechnology6, 1197-1202 (1988)), nucleic acid sequence-based amplification (or“NASBA”) (see R. Lewis, Genetic Engineering News 12 (9), 1 (1992)), therepair chain reaction (or “RCR”) (see R. Lewis, supra), and boomerangDNA amplification (or “BDA”) (see R. Lewis, supra). Polymerase chainreaction is generally preferred.

“Amplification” is a special case of nucleic acid replication involvingtemplate specificity. It is to be contrasted with non-specific templatereplication (i.e., replication that is template-dependent but notdependent on a specific template). Template specificity is heredistinguished from fidelity of replication (i.e., synthesis of theproper polynucleotide sequence) and nucleotide (ribo- or deoxyribo-)specificity. Template specificity is frequently described in terms of“target” specificity. Target sequences are “targets” in the sense thatthey are sought to be sorted out from other nucleic acid. Amplificationtechniques were designed primarily for this sorting out.

As used herein, the term “polymerase chain reaction” and “PCR” generallyrefers to the method for increasing the concentration of a segment of atarget sequence in a mixture of genomic DNA without cloning orpurification (U.S. Pat. Nos. 4,683,195; 4,683,202; and 4,965,188; hereinincorporated by reference). This process for amplifying the targetsequence comprises introducing an excess of two oligonucleotide primersto the DNA mixture containing the desired target sequence, followed by aprecise sequence of thermal cycling in the presence of a DNA polymerase.The two primers are complementary to their respective strands of thedouble stranded target sequence. To effect amplification, the mixture isdenatured and the primers then annealed to their complementary sequenceswithin the target molecule. Following annealing, the primers areextended with a polymerase so as to form a new pair of complementarystrands. The steps of denaturation, primer annealing and polymeraseextension can be repeated many times (i.e., denaturation, annealing andextension constitute one “cycle”; there can be numerous “cycles”) toobtain a high concentration of an amplified segment of the desiredtarget sequence. The length of the amplified segment of the desiredtarget sequence is determined by the relative positions of the primerswith respect to each other, and therefore, this length is a controllableparameter. By virtue of the repeating aspect of the process, the methodis referred to as the “polymerase chain reaction” (hereinafter “PCR”).Because the desired amplified segments of the target sequence become thepredominant sequences (in terms of concentration) in the mixture, theyare said to be “PCR amplified.

The term “plurality” is used herein to mean two or more, for example,three, four, five or more, including ten, twenty, fifty or morepolynucleotides, nucleic acid probes, and the like.

The term “reverse-transcriptase” or “RT-PCR” refers to a type of PCRwhere the starting material is mRNA. The starting mRNA is enzymaticallyconverted to complementary DNA or “cDNA” using a reverse transcriptaseenzyme. The cDNA is then used as a “template” for a “PCR” reaction.

In an embodiment, the amplification reaction is quantified. In otherembodiments, the amplification reaction is quantitated using a signatureprofile, in which the signature profile is selected from the groupconsisting of a melting temperature or a fluorescence signature profile.

The nucleic acid molecule of embodiments of the disclosure, or segmentsthereof, can be used as primers for PCR amplification. In performing PCRamplification, a certain degree of mismatch can be tolerated betweenprimer and template. Therefore, mutations, deletions, and insertions(especially additions of nucleotides to the 5′ or 3′ end) of theexemplified primers fall within the scope of the subject disclosure.Mutations, insertions, and deletions can be produced in a given primerby methods known to an ordinarily skilled artisan.

Molecular Beacons have been described for use in sequence detection.Briefly, a FRET oligonucleotide probe is designed that overlaps theflanking genomic and insert DNA junction. The unique structure of theFRET probe results in it containing a secondary structure that keeps thefluorescent and quenching moieties in close proximity. The FRET probeand PCR primers (one primer in the insert DNA sequence and one in theflanking genomic sequence) are cycled in the presence of a thermostablepolymerase and dNTPs. Following successful PCR amplification,hybridization of the FRET probe(s) to the target sequence results in theremoval of the probe secondary structure and spatial separation of thefluorescent and quenching moieties. A fluorescent signal indicates thepresence of the flanking genomic/transgene insert sequence due tosuccessful amplification and hybridization. Such a molecular beaconassay for detection of as an amplification reaction is an embodiment ofthe subject disclosure.

Hydrolysis probe assay, otherwise known as TAQMAN® (Life Technologies,Foster City, Calif.), is a method of detecting and quantifying thepresence of a DNA sequence. Briefly, a FRET oligonucleotide probe isdesigned with one oligo within the transgene and one in the flankinggenomic sequence for event-specific detection. The FRET probe and PCRprimers (one primer in the insert DNA sequence and one in the flankinggenomic sequence) are cycled in the presence of a thermostablepolymerase and dNTPs. Hybridization of the FRET probe results incleavage and release of the fluorescent moiety away from the quenchingmoiety on the FRET probe. A fluorescent signal indicates the presence ofthe flanking/transgene insert sequence due to successful amplificationand hybridization. Such a hydrolysis probe assay for detection of as anamplification reaction is an embodiment of the subject disclosure.

KASPar assays are a method of detecting and quantifying the presence ofa DNA sequence. Briefly, the genomic DNA sample comprising the targetedgenomic locus is screened using a polymerase chain reaction (PCR) basedassay known as a KASPar® assay system. The KASPar® assay used in thepractice of the subject disclosure can utilize a KASPar® PCR assaymixture which contains multiple primers. The primers used in the PCRassay mixture can comprise at least one forward primers and at least onereverse primer. The forward primer contains a sequence corresponding toa specific region of the donor DNA polynucleotide, and the reverseprimer contains a sequence corresponding to a specific region of thegenomic sequence. In addition, the primers used in the PCR assay mixturecan comprise at least one forward primers and at least one reverseprimer. For example, the KASPar® PCR assay mixture can use two forwardprimers corresponding to two different alleles and one reverse primer.One of the forward primers contains a sequence corresponding to specificregion of the endogenous genomic sequence. The second forward primercontains a sequence corresponding to a specific region of the donor DNApolynucleotide. The reverse primer contains a sequence corresponding toa specific region of the genomic sequence. Such a KASPar® assay fordetection of an amplification reaction is an embodiment of the subjectdisclosure.

In some embodiments the fluorescent signal or fluorescent dye isselected from the group consisting of a HEX fluorescent dye, a FAMfluorescent dye, a JOE fluorescent dye, a TET fluorescent dye, a Cy 3fluorescent dye, a Cy 3.5 fluorescent dye, a Cy 5 fluorescent dye, a Cy5.5 fluorescent dye, a Cy 7 fluorescent dye, and a ROX fluorescent dye.

In other embodiments the amplification reaction is run using suitablesecond fluorescent DNA dyes that are capable of staining cellular DNA ata concentration range detectable by flow cytometry, and have afluorescent emission spectrum which is detectable by a real timethermocycler. It should be appreciated by those of ordinary skill in theart that other nucleic acid dyes are known and are continually beingidentified. Any suitable nucleic acid dye with appropriate excitationand emission spectra can be employed, such as YO-PRO-1®, SYTOX Green®,SYBR Green I®, SYTO11®, SYTO12®, SYTO13®, BOBO®, YOYO®, and TOTO®. inone embodiment, a second fluorescent DNA dye is SYTO13® used at lessthan 10 μM, less than 4 μM, or less than 2.7 μM.

Embodiments of the present invention are further defined in thefollowing Examples. It should be understood that these Examples aregiven by way of illustration only. From the above discussion and theseExamples, one skilled in the art can ascertain the essentialcharacteristics of this invention, and without departing from the spiritand scope thereof, can make various changes and modifications of theembodiments of the invention to adapt it to various usages andconditions. Thus, various modifications of the embodiments of theinvention, in addition to those shown and described herein, will beapparent to those skilled in the art from the foregoing description.Such modifications are also intended to fall within the scope of theappended claims. The following is provided by way of illustration andnot intended to limit the scope of the invention.

EXAMPLES Example 1 Analysis of Targeted Loci in Maize Callus

Genomic Loci Targeting: The genomic locus for corn event DAS-59132 thatwas previously disclosed in WO2009100188 METHODS FOR DETECTION OF CORNEVENT DAS-59132, herein incorporated by its entirety, was targeted usinga zinc finger nuclease designed to specifically bind and cleave thegenomic DNA which makes up this event. The resulting transformants weremaintained until an analysis to identify and characterize the disruptionof the genomic loci within specific events via an amplification reactioncould be completed.

The zinc finger proteins directed against DNA sequences which comprisethe genomic locus for DAS-59132 were designed as previously described.See, e.g., Urnov et al. (2005) Nature 435:646-651. The DAS-59132 zincfinger designs were incorporated into vectors encoding a protein havingat least one finger with a CCHC structure. See, U.S. Patent PublicationNo. 2008/0182332. In particular, the last finger in each protein had aCCHC backbone for the recognition helix. The non-canonical zincfinger-encoding sequences were fused to the nuclease domain of the typeIIS restriction enzyme, FokI (amino acids 384-579 of the sequence of Wahet al. (1998) Proc. Natl. Acad. Sci. USA 95:10564-10569) via a fouramino acid linker and an opaque-2 nuclear localization signal derivedfrom Zea mays to form DAS-59132 zinc-finger nucleases (ZFNs). Expressionof the fusion proteins in a bicistronic expression construct utilizing a2A ribosomal stuttering signal as described in Shukla et al. (2009)Nature 459:437-441 was driven by a relatively strong, constitutive andectopic promoter such as the CsVMV promoter.

The optimal ZFNs were verified for cleavage activity using a buddingyeast based system previously shown to identify active nucleases. See,e.g., U.S. Patent Publication No. 20090111119; Doyon et al. (2008) NatBiotechnol. 26:702-708; Geurts et al. (2009) Science 325:433. Of thenumerous ZFNs that were designed, produced and tested to bind to theputative DAS-59132 genomic polynucleotide target sites, preferred ZFNswere identified as having in vivo activity at high levels, and selectedfor further experimentation. These ZFNs were characterized as beingcapable of efficiently binding and cleaving the DAS-59132 genomicpolynucleotide target sites in planta.

Plasmid vectors containing ZFN expression constructs of the exemplaryzinc finger nucleases, which were identified using the yeast assay, weredesigned and completed using skills and techniques commonly known in theart. Next, the opaque-2 nuclear localization signal::zinc fingernuclease fusion sequence was paired with the complementary opaque-2nuclear localization signal::zinc finger nuclease fusion sequence. Assuch, each construct consisted of a single open reading frame comprisedof two opaque-2 nuclear localization signal::zinc finger nuclease fusionsequences separated by the 2A sequence from Thosea asigna virus (Mattionet al. (1996) J. Virol. 70:8124-8127). Expression of the ZFN codingsequence was driven by the highly expressing constitutive Zea maysUbiquitin 1 Promoter (Christensen et al. (1992) Plant Mol. Biol.18(4):675-89) and flanked by the Zea mays Per 5 3′ polyA untranslatedregion (U.S. Pat. No. 6,699,984).

A donor construct was designed to integrate into the ZFN cleaved genomicDNA of the DAS-59132 genomic locus. This single gene expression cassetteis driven by the Rice Actin1 promoter (Os Act1 promoter):: thephosphinothricin acetyl transferase coding sequence (PAT; U.S. Pat. No.7,838,733):: and is terminated by the Zea mays lipase 3′ untranslatedregion (ZmLip 3′UTR). In addition, the donor plasmid was designed with 1kB sequence (homology arms) on either end of the target PAT gene that ishomologous to sequence on either end of the ZFN cut site in theDAS-59132 genomic locus. The homology arms served as the substrate thatthe homologous recombination machinery used to insert the transgene intothe genomic ZFN cut site. The various gene elements were assembled in ahigh copy number pUC based plasmid.

Targeted Integration: Transgenic events were targeted to the endogenousgenomic locus of DAS-59132. Constructs as described previously includethe donor sequence (pDAB107855) and DAS-59132 ZFN 6 (pDAB105906).Co-transformation of these two plasmids resulted in 854 transgenic PATevents that were screened with the method for identifying the presenceof a donor DNA polynucleotide within a targeted genomic locus of thesubject disclosure.

Maize callus cells, consisting of 12 mL of packed cell volume (PCV) froma previously cryo-preserved cell line plus 28 mL of conditioned mediumwas subcultured into 80 mL of GN6 liquid medium in a 500 mL Erlenmeyerflask, and placed on a shaker at 125 rpm at 28° C. This step wasrepeated two times using the same cell line, such that a total of 36 mLPCV was distributed across three flasks. After 24 hours, the GN6 liquidmedia was removed and replaced with 72 mL GN6 S/M osmotic medium. Theflask was incubated in the dark for 30-35 minutes at 28° C. withmoderate agitation (125 rpm). During the incubation period, a 50 mg/mLsuspension of silicon carbide WHISKERS™ (Advanced Composite Materials,LLC, Greer, S.C.) was prepared by adding 8.1 mL of GN6 S/M liquid mediumto 405 mg of sterile, silicon carbide WHISKERS™.

Following incubation in GN6 S/M osmotic medium, the contents of eachflask were pooled into a 250 mL centrifuge bottle. After all cells inthe flask settled to the bottom, the content volume in excess ofapproximately 14 mL of GN6 S/M liquid was drawn off and collected in asterile 1-L flask for future use. The pre-wetted suspension of WHISKERS™was mixed at maximum speed on a vortex for 60 seconds, and then added tothe centrifuge bottle.

In this example, pDAB 107855 (donor sequence) and pDAB 105906 (ZFN)plasmid DNA were added to each bottle. Once the plasmid DNA was added,the bottle was immediately placed in a modified RED DEVIL 5400™commercial paint mixer (Red Devil Equipment Co., Plymouth, Minn.), andagitated for 10 seconds. Following agitation, the cocktail of cells,media, WHISKERS™ and plasmid DNA were added to the contents of a 1 Lflask along with 125 mL fresh GN6 liquid medium to reduce theosmoticant. The cells were allowed to recover on a shaker set at 125 rpmfor 2 hours. 6 mL of dispersed suspension was filtered onto Whatman #4filter paper (5.5 cm) using a glass cell collector unit connected to ahouse vacuum line such that 60 filters were obtained per bottle. Filterswere placed onto 60×20 mm plates of GN6 solid medium and cultured at 28°C. under dark conditions for 1 week.

One week post-DNA delivery, filter papers were transferred to 60×20 mmplates of GN6 (1H) selection medium containing a selective agent. Theseselection plates were incubated at 28° C. for one week in the dark.Following 1 week of selection in the dark, the tissue was embedded ontofresh media by scraping ½ the cells from each plate into a tubecontaining 3.0 mL of GN6 agarose medium held at 37-38° C.

The agarose/tissue mixture was broken up with a spatula and,subsequently, 3 mL of agarose/tissue mixture was evenly poured onto thesurface of a 100×25 mm petri dish containing GN6 (1H) medium. Thisprocess was repeated for both halves of each plate. Once all the tissuewas embedded, plates incubated at 28° C. under dark conditions for up to10 weeks. Putatively transformed isolates that grew under theseselection conditions were removed from the embedded plates andtransferred to fresh selection medium in 60×20 mm plates. If sustainedgrowth was evident after approximately 2 weeks, an event was deemed tobe resistant to the applied herbicide (selective agent) and an aliquotof cells was subsequently harvested for genotype analysis. In thisexample, a large number of events were recovered from the treatedbottles. These events were advance for molecular analysis to confirm theintegration of a transgene within a genomic locus of Corn EventDAS-59132.

DNA Extraction: Callus tissue samples were collected in 96-wellcollection plates (Qiagen, Valencia, Calif.) and then lyophilized for 48hours. Tissue disruption was performed with a KLECKO™ tissue pulverizer(Garcia Manufacturing, Visalia, Calif.) in BIOSPRINT96 AP1™ lysis buffer(Qiagen) with one stainless steel bead. Following tissue maceration,genomic DNA was isolated in high throughput format using theBIOSPRINT96™ plant kit (Qiagen) using the BIOSPRINT96™ extraction robot(Qiagen). A sample of genomic DNA was then diluted to 2 ng/μl prior tosetting up the qPCR reactions to achieve appropriate Cp (quantificationcycle) scores which resulted in the production of a signature profile.

DAS-59132 Locus Disruption Assay: WHISKERS™ mediated transformation ofHi-II callus cells with the DAS-59132-ZFN and donor plasmid resulted intargeted and random transgene insertions. To distinguish randominsertion events from the targeted event populations, all 854 eventsgenerated were initially screened using a locus disruption assay. Thisassay determined whether the ZFN binding site within the locus remainsintact or had been disrupted through ZFN cleavage or donor insertion.Indication of a disruption within the genomic loci is initial evidencethat the ZFN has cleaved the endogenous DAS-59132 target locus andindicates targeted insertion of the donor DNA molecule. Primers weredesigned to amplify the endogenous target region that contains the ZFNrecognition sites, and samples were set up to be analyzed by qPCR.Amplification of the intact region, indicative of an untargeted event,resulted in a 140 base pair amplicon measured as a detectable qPCRsignal. Successful targeted integration of the donor molecule results indisruption of the detectable qPCR signal and is shown as a lower overallsignal compared to control.

The DAS-59132 locus disruption assay was performed by real-time PCRusing the LIGHTCYCLER®480 system (Roche Applied Science, Indianapolis,Ind.). Assays were designed to monitor the DAS-59132 ZFN (25716/25717)binding sequences at the DAS-59132 locus (and the internal referencegene IVF (Genbank Acc No: U16123.1|ZMU16123) using LIGHTCYCLER® ProbeDesign Software 2.0. For amplification, LIGHTCYCLER®480 Probes Mastermix (Roche Applied Science, Indianapolis, Ind.) was prepared at 1× finalconcentration in a 10 μL volume multiplex reaction containing 0.4 μM ofeach primer and 0.2 μM of each probe (Table 1). A two step amplificationreaction was performed with an extension at 55° C. for 30 seconds withfluorescence acquisition. Analysis for the disruption assay wasperformed using target to reference ratio.

TABLE 1 Oligonucleotide Primer and Probe Sequences for DAS-59132 Locus Disruption Assay. Primer SEQ ID  Detec- Name NO:Sequence tion MAS604 SEQ ID  ACACGGCACACACGGCGACATTCA — NO: 1  MAS606SEQ ID  AGGGCAGTGGCCAGTGTTCCTGTG — NO: 2  UPL 69 — Roche Sequence FAMIVF-Taq  SEQ ID  TGGCGGACGACGACTTGT — NO: 3  IVR-Taq  SEQ ID AAAGTTTGGAGGCTGCCGT — NO: 4  IV-Probe SEQ ID  CGAGCAGACCGCCGTGTACTTCTACCHEX NO: 5 

The 854 events generated from precision transformation were screenedwith the disruption assay, and scored as disrupted based on asignificant drop in the target to reference signal. The resultsindicated that 63 of the 854 events assayed had a disrupted signal atthe DAS-59132 locus, indicative of targeted gene insertion (FIG. 2).Despite the quantitative measurement of a disrupted locus that thedisruption assay provides, this assay cannot resolve targeted insertionsfrom mutations at the cleavage site resulting from error prone breakrepair. The development of a secondary In-Out PCR assay for screeningevents in parallel to ensure robust and accurate evaluation of allevents was developed.

DAS-59132 Locus In-Out PCR Assay: The events screened by the disruptionassay were also screened by a locus specific end-point PCR assay at theDAS-59132 locus. One oligonucleotide primer is designed to anneal to aregion of target genomic DNA outside the ZFN cleavage site and a secondoligonucleotide primer is designed to anneal only to the transgeneregion of the donor DNA. The primers are designed to analyze the 5′ and3′ insert DNA junction regions of the target site at the DAS-59132locus. Many of the events generated from transformation are randominsertions and so are not amplified during the In-Out PCR, as the donorsequence is not in proximity to the target sequence (FIG. 3A). As theprimers are designed to only amplify regions of donor DNA and genomicDNA that are inserted in the targeted region, amplification is a resultof a targeted transgene event (FIG. 3B). This PCR analysis, called“In-Out” PCR, ensures that a complete picture of targeted gene insertionis obtained, as the primers are designed to target both the targetsequence and inserted donor sequence and both the 5′ and 3′ junctionsare analyzed. Following PCR, amplified samples are analyzed byelectrophoresis and samples with transgene integration at the targetsite in the DAS-59132 locus result in amplification of two bands at 2 kband 1.5 kb. This is indicative of integrated target sequence at the 5′and 3′ junctions of the transgene.

In-Out PCR amplification reactions were conducted using a Takara Ex TaqHS Kit™ (Clontech Laboratories, Inc., Mountain View, Calif.). Each PCRreaction was carried out in 15 or 20 μL final volume, which contained1×Ex Taq buffer, 200 nM of forward and reverse primers, 10 to 20 ng ofgenomic DNA template, and a final concentration of 0.05 unit/μL Ex TaqHS polymerase. For real-time In-Out PCR, a SYTO13® dye from Invitrogen(Grand Island, N.Y.) was included in the PCR reaction mix at a finalconcentration of 4 μM or 2.67 μM. Initially the SYTO13® greenfluorescent dye was used per manufacturer recommended concentrations asit had been previously shown to increase overall assay sensitivity andexhibit low inhibition of polymerase activity. This dye resulted in astronger signal and more consistent results, but the high-throughputsystem still had limitations. Background fluorescence continued to beproblematic, as primer-dimer formations or non-specific primer annealing(from impure primers) were generating false positive signals. As such,the concentrations which were used in the assay were lower from 10 μM to4 μM or 2.67 μM. The reduction in concentration of the dye resulted in asignature profile that provided reliable detection and quantitation ofthe PCR assay.

Real-time In-Out PCR was performed on an ABI VIIA7 PCR SYSTEM™ (LifeTechnologies Corporation, Carlsbad, Calif.). After initial denaturing,the amplification program contained 40 cycles of 98° C. for 10 sec, 66°C. for 30 sec and 68° C. for 2 min with fluorescence acquisition beforea melting temperature analysis program. Following the amplificationstep, the reaction was kept at 65° C. for 30 sec and 72° C. for 10 min,and finally held at 4° C. Both direct fluorescence signals and meltingtemperature profiles were used for sample analysis. Positive samplesidentified on the real-time system were further confirmed using astandard gel shift assay.

TABLE 2 Primer and Probe Sequences for  DAS-59132 Locus In Out Assay.Primer SEQ ID Name NO: Primer Sequence 5′ E32-5F3 SEQ ID GAAGGCAAAACGAATATAAG Junction  NO: 6 TGCATTCGG Sequence E32- SEQ ID TCGTGGATAGCACTTTGGGCT OLP-R1 NO: 7 3′ E32- SEQ ID  TCTACAGTGAACTTTAGGACAJunction  OLP-F3 NO: 8 GAGCCA Sequence E32-3R2 SEQ ID GCCCTTACAGTTCATGGGCG NO: 9

In an effort to differentiate targeted insert PCR amplicons from falsepositives, a protocol was designed to assign a signature profile toevery PCR product generated. Melting temperature profiles of the PCRamplicons were compared to a positive control, and matching curvesidentified positive In-Out PCR products (FIGS. 4A and 4B). Correlatingthe In-Out PCR analysis using a signature profile that comprises amelting temperature profile of both the both the 3′ and 5′ ends is anovel analytical methodology that generates greater confidence inidentifying a targeted donor DNA polynucleotide insertion event within agenomic locus.

The results of the disruption assay and the DAS-59132 locus In-Out PCRassay were further confirmed via Southern blotting and sequencing(standard of Next Generation Sequencing).

The novel assay resulted in a robust analytical process to identifytargeted donor DNA polynucleotide insertion events at a ZFN cleavagesite in maize. The endogenous genomic loci was successfully targeted andthe targeted events were efficiently identified using the novel assay. Atotal of 854 samples were submitted for the analysis using the disclosedassay. The disruption assay was performed on all of the putative eventswith 63 of the events showing disruption. The In-Out PCR was performedon all of the events, and 8 positive events were identified. As aresult, there were a total of 8 events that were confirmed to betargeted inserts (FIG. 5).

Example 2 Analysis of Targeted Loci in Maize Plants

Maize transgenic B104 embryos were generated, wherein the DAS-59132locus was targeted via the Zinc Finger Nuclease construct, pDAB105906,and a donor construct, pDAB 104179. These constructs were transformedinto the plant tissue using a biolistic transformation method asdescribed in Example 7 of US Patent Application No. 2011/0191899, hereinincorporated by reference in its entirety. Putatively transformedembryos were identified via selection of the herbicide phosphinothricin.

The putatively identified transgenic embryos were analyzed using thedisruption assay to identify events which contained the presence of adonor DNA polynucleotide inserted within a targeted genomic locus. TheZFN disruption assay was completed using the protocols and reagentsdescribed above. In the events that were not targeted or disrupted, atarget to reference ratio in the 0.4 to 0.6 range was observed; forsamples that were disrupted or targeted a range from 0.2 to 0.35 (plateto plate variation) was reported (FIG. 6). The targeted events which didnot produce an amplicon resulted in a lower quantity of amplifiedproduct, as depicted in the graph.

Next, locus specific In-Out PCR was completed using the protocol andreagents described above. The results of the In-Out PCR identifiedspecific events which contained a transgene insertion within theDAS-59132 locus.

A total of 1,223 sample events were submitted for the analysis. Thedisruption assay was completed on every event, and identified 85 of theevents showing disruption. The In-Out PCR was completed on all 1,223sample events and identified 11 events that were positive and 2 partialpositive events. Southern blotting and sequencing was completed toconfirm that the identified events comprised full transgene insertionswithin the DAS-59132 genomic locus.

Example 3 Analysis of Targeted Loci in Maize Plants

Maize transgenic B104 embryos were generated, wherein an engineeredlanding pad locus (US Patent Application No. 2011/0191899, hereinincorporated by reference in its entirety) was targeted via the ZincFinger Nuclease and a donor construct were transformed into the planttissue using biolistics. Multiple transformations were completed totarget a donor construct within the engineered landing pad locus. Thefirst series of transformations were completed using the pDAB109714donor construct and the pDAB 105941 Zinc Finger Nuclease construct. Asecond series of transformations were completed using the pDAB 109715donor construct and the pDAB 105943 Zinc Finger Nuclease construct. Athird series of transformations were completed using the pDAB 109716donor construct and the pDAB 105942 Zinc Finger Nuclease construct. Thefinal series of transformations were completed using the pDAB109717donor construct and the pDAB 105945 Zinc Finger Nuclease construct.Putatively transformed embryos were identified via selection of theherbicide phosphinothricin.

ELP Locus Disruption Assay: Primers were designed to amplify theendogenous target region that contains the ZFN recognition sites, andsamples were set up to be analyzed by qPCR. Amplification of the intactregion, indicative of an untargeted event, resulted in a 193 base pairamplicon measured as a detectable qPCR signal. Successful targetedintegration of the donor molecules within the respective ELP eventresults in disruption of the detectable qPCR signal and is shown as alower overall signal compared to control.

The ELP locus disruption assay was performed by real-time PCR using theLIGHTCYCLER®480 system (Roche Applied Science, Indianapolis, Ind.).Assays were designed to monitor the ELP ZFN binding sequences at the ELPlocus, and the internal reference gene IVF using LIGHTCYCLER® ProbeDesign Software 2.0). For amplification, LIGHTCYCLER®480 Probes Mastermix (Roche Applied Science, Indianapolis, Ind.) was prepared at 1× finalconcentration in a 10 μL volume multiplex reaction containing 0.4 μM ofeach primer and 0.2 μM of each probe (Table 3). A two step amplificationreaction was performed with an extension at 55° C. for 30 seconds withfluorescence acquisition. Analysis for the disruption assay wasperformed using target to reference ratio.

TABLE 3 Primer and Probe Sequences for ELP Locus Disruption Assay. The ELP1 and ELP2 reactions were multiplexed with the IVF  primers and probe, the sequences  of which are described in Table 1.SEQ  Primer ID Detec- Name NO: Sequence tion ELP1 MAS622 SEQ TAGGAGTTCTCTTTTATGCCACCC — ID NO: 10 MAS621 SEQ CCTTGGGATTTCAGTTGGTAGGTT — ID NO: 11 UPL69 — Roche Sequence FAM ELP2MAS617 SEQ  TGGGTAGGAGGACACCAAAGATGA — ID NO: 12 MAS 618 SEQ CCATTGGATTATTGAAAACTGGCAG — ID NO: 13 UPL122 — Roche Sequence FAM

The 1738 events generated from precision transformation were screenedwith the disruption assay, and scored as disrupted based on asignificant drop in the target to reference signal. The resultsindicated that 158 of the 1738 events assayed had a disrupted signal atthe ELP locus, indicative of targeted gene insertion (FIG. 7). Despitethe quantitative measurement of a disrupted locus that the disruptionassay provides, this assay does not resolve targeted insertions frommutations at the cleavage site resulting from error prone break repair.Therefore, the development of a secondary In-Out PCR assay for screeningevents in parallel to ensure robust and accurate evaluation of allevents was developed.

ELP Loci In-Out PCR Assay: The events screened by the disruption assaywere also screened by a newly-developed locus specific end-point PCRassay at the ELP loci. One primer was designed to anneal to a region oftarget genomic DNA outside the ZFN cleavage site and a second primer wasdesigned to anneal only to the transgene region of the donor DNA. Theprimers were designed to analyze the 5′ and 3′ regions of the targetsite at the ELP locus. As the primers were designed to only amplifyregions of donor DNA and genomic DNA that are inserted in the targetedregion, amplification is a result of a targeted transgene event, both ofthe 5′ and 3′ junctions were analyzed.

In-Out PCR amplification reactions were conducted using a TAKARA EX TAQHS KIT™ (Clontech Laboratories, Inc., Mountain View, Calif.). Each PCRreaction was carried out in 15 or 20 μL final volume, which contained1×Ex Taq buffer, 200 nM of forward and reverse primers, 10 to 20 ng ofgenomic DNA template, and a final concentration of 0.05 unit/μL Ex TaqHS polymerase. For real-time In-Out PCR, a SYTO13® dye (Invitrogen,Carlsbad, Calif.) was included in the PCR reaction mix at a finalconcentration of 4 μM or 2.67 μM.

Real-time In-Out PCR was performed on an ABI VIIA7 PCR SYSTEM™ (LifeTechnologies Corporation, Carlsbad, Calif.). After initial denaturing,the amplification program contained 40 cycles of 98° C. for 10 sec, 66°C. for 30 sec and 68° C. for 2 min with fluorescence acquisition beforea melting temperature analysis program. Following that, the reaction waskept at 65° C. for 30 sec and 72° C. for 10 min, and finally held at 4°C. Both direct fluorescence signals and melting temperature profileswere used for sample analysis. Positive samples identified on thereal-time system were further confirmed using a standard gel shiftassay.

TABLE 4 Primer and Probe Sequences for  ELP Locus In Out Assay. SEQ Primer ID  Name NO: Primer Sequence 5′ ELP1- SEQ AGA CCT ACC ACC CAT TAG GGC Junc- PriF1 ID  tion  NO: 14 Se- OsAct- SEQ TCG TGG ATA GCA CTT TGG GCT quence PriR3 ID  NO: 15 3′ AAD1- SEQ CTT GAC TCG CAC CAC AGT TGG Junc- PriF1 ID  tion  NO: 16 Se- ELP2- SEQ GAT GGT GGT TAT GAC AGG CTC CT quence PriR1 ID  NO: 17

In an effort to identify PCR amplicons from false positives, a protocolwas designed to assign a signature profile to every PCR productgenerated. Melting temperature profiles of the PCR amplicons werecompared to a positive control, and matching curves identified positiveIn-Out PCR products. Correlating the In-Out PCR analysis using asignature profile that comprises a melting temperature profile of boththe both the 3′ and 5′ ends is an analytical methodology that generatesgreater confidence in identifying a targeted transgene insertion event.

A total of 1738 PAT positive samples were submitted for this project.The disruption assay and the In-Out PCR was performed on all events. Theresults indicated that 158/1738 events were positive for disruption, and46/1738 of these events were positive for both the 3′ and 5′ In-Out PCRamplification reactions.

Example 4 Analysis of Targeted Loci in Maize Plants

Zea mays c.v. B104 plants were transformed with an engineered landingpad gene construct (pDAB105817 or pDAB105818) as previously described inUS Patent Application No. 2011/0191899. Transformed maize plants wereobtained and confirmed to contain the ELP. Four ELP maize lines;105817[1]-015.Sx001.Sx011, 105818[1]-269.Sx001.Sx008,105818[1]-271.Sx001.Sx005, and 105818[2]-388.Sx001.Sx008 were crossedwith Zea mays c.v. B104 to produce as hemizygotes. The resulting progenyplants were co-transformed with either eZFN plasmid pDAB105941 encodingeZFN1 and corresponding donor plasmid pDAB104182 or eZFN pDAB 105948encoding eZFN8 and donor pDAB 104183. The transformations were completedvia bombardment of isolated embryos that were shot one time using aPDS-1000 (Bio-Rad) per manufacturer's specifications. A total of 20,896embryos (about 5000 embryos for each target line) were bombarded andselected for Bialaphos resistance: 12,404 were co-bombarded with pDAB105941 and its corresponding donor, and 8,492 embryos were co-bombardedwith pDAB 105948 and its corresponding donor.

Following bombardment, embryos were cultured in media and grown intoplantlets. Transgenic events were identified via a qPCR that wasdeveloped to screen and detect the presence of the pat transgene. Thecopy number were determined by comparison of Target/Reference(Invertase) values for unknown samples (output by the LightCycler 480™)to Target/Reference values of known copy number standards (1-Copy: hemi,2-Copy: homo). A total of 614 regenerated plants survived, and 354 (or58%) of the plants were identified as positive for the presence of thepat gene and retained for further analysis.

ELP Locus Disruption Assay: A ZFN disruption assay was designed tomonitor the changes in the integrated ELP. In a non-targeted ELP, PCRprimers MAS621 and MAS622 (Table 5) amplified a 214 bp productencompassing the eZFN binding sites. Integration into (or modificationof) the eZFN binding site at the ELP locus would disrupt amplificationusing qPCR with low extension time resulting in either no signal orsignificantly lower signal being produced in the qPCR reaction. Acontrol qPCR reaction for the amplification of the Invertase gene wasalso included as an internal control reference.

For amplification, LightCycler®480 Probes Master mix (Roche AppliedScience, Indianapolis, Ind.) was prepared at 1× final concentration in a10 μL volume multiplex reaction containing 0.4 μM of each primer and 0.2μM of each probe. A three step amplification reaction was performed with10 seconds at 95° C. for denaturation, 35 seconds at 60° C. forannealing and 1 second at 72° C. with fluorescence acquisition. The FAMfluorescent moiety was excited at an optical density of 465/510 nm andHEX at 533/580 nm. The copy number was determined by comparison ofTarget/Reference (Invertase) values for unknown samples (output by theLightCycler 480™) to Target/Reference values of known single copyhemizygotes.

To distinguish random insertion events from ELP-targeted events, the 354samples that were identified via the pat qPCR screening were furtheranalyzed using the disruption assay. The ELP disruption assay wasdesigned to monitor the large insertions/deletions in the eZFN cleavagesite. In a non-targeted ELP, a 214 bp PCR product encompassing the eZFNbinding sites is amplified and generates strong fluorescent signals.Integration into (or modification of) the eZFN binding site woulddisrupt amplification and result in either no signal or significantlylower levels of fluorescent signal produced via the PCR reaction. Out ofthe 354 pat positive events, about 8% (28 events) appeared to bedisrupted (FIG. 8 b), indicating potential targeting.

TABLE 5 Primer and probe used for disruption assay. Primer SEQ ID  NameNO: Sequence MAS621 SEQ ID  CCTTGGGATTTC Dis- NO: 18 AGTTGGTAGGTTruption UPL67 — Roche Sequence assay MAS622 SEQ ID  TAGGAGTTCTCT NO: 19TTTATGCCACCC InvertaseF SEQ ID  TGGCGGACGACGA Inver- NO: 20 CTTGT taseInvertaseProbe SEQ ID  Hex-CGAGCAGAC Refer- NO: 21 CGCCGTGTACTT enceInvertaseR SEQ ID  AAAGTTTGGAGGC gene NO: 22 TGCCGT

In-Out PCR: The disruption assay identified changes in the eZFN bindingregion on the ELP. Any variation in the primer binding sites or randominsertions in the locus are possible indications of disruption to thelocus that is caused by ZFN cleavage. Next, an In-Out PCR was used tovalidate the presence of a donor insertion within the ELP locus. Theboth ends of the junction sequences comprising the target locus and thedonor. One primer was designed to anneal to a pre-integrated ELP regiononly present in the target lines and a second primer was designed toanneal only to donor DNA sequences that are only present within thedonor construct. Amplification via PCR indicated a result of targetingof the donor within the ELP genomic locus. Any events generated fromrandom insertions do not produce a PCR amplification.

For the donor/ELP 5′ junction In-Out PCR, the forward primer wasdesigned to bind the OsAct1 region (5OsF3, SEQ ID NO:23ATTTCACTTTGGGCCACCTT) of the donor insert, while the reverse primer(5R3, SEQ ID NO:24 AGGCTCCGTTTAAACTTGCTG) was designed to bind the 193bp sequence unique to the target line in the ELP Left Arm. For the 3′donor/ELP locus junction In-Out PCR, the forward primer (3PAF1, SEQ IDNO:25 ATGGTGGATGGCATGATGTT) was designed to bind the in the PATv6 regionof the donor insert, and the reverse primer (3R1, SEQ ID NO:26TGGAGGTTGACCATGCTAGG) was designed to bind the 192 bp sequence unique tothe target line in the ELP Right Arm. Amplification resulted only whenthe forward and reverse primers bound to sequences in close proximity ofone another. Random integration of the donor sequences was notdetectable using the above described PCR primers. To increase thethroughput of the In-Out PCR detection, the melting curve analysis wascompleted with the green fluorescent nucleic acid stain, SYTO13®. ThePCR amplification was performed on LightCycler 480™ real time PCR systemin a 15 μl reaction containing 0.75 units of TaKaRa Ex Taq™ DNApolymerase (Takara Bio Inc., Shiga, Japan), 200 nM of dNTP, 200 nM eachof forward and reverse primers, 2.67 μM of SYTO13® and 10 ng of genomicDNA. Amplification started with a 2 min. denaturing cycle at 95° C.,then 30 cycles of 98° C. for 20 seconds, 60° C. for 30 seconds and 68°C. for 90 seconds, followed by melting curve analysis at 97° C. with0.11° C./s ramp speed.

The results of the PCR reaction resulted in the analysis of both the 5′and 3′ flanking ends. A donor targeted ELP locus event results in a 1.1kb fragment for the 5′ In-Out PCR reaction and a 1.4 kb fragment for the3′ In-Out PCR reaction. The results of the PCR reactions were determinedvia melting curve analysis, which produces different graphical resultsdepending on the length and composition of the amplicon (FIG. 9). ShortPCR amplicons usually display a single-peak melting temperature (Tm)curve that appears as a flat line and produces low-levels of fluorescentsignal when displayed graphically. But for long amplicons (i.e., 1-2 kbfragments), the Tm profile appears more complex, and results in multiplepeaks (depending on local sub-sequences of the amplicons) and produceshigh-levels of fluorescent signal. Five events were identified to bepositive for both the 5′ and 3′ PCR reactions. Each of the four targetlines produced an event comprising a donor inserted within the ELPgenomic locus. All of the In-Out PCR positives were run on gel wherethey only showed one band of PCR amplicon, as expected.

Molecular Confirmation: Additional molecular detection methods werecompleted to confirm that the results of the disruption assay and In-OutPCR reactions were not false positives. A Southern blot analysis andsequencing of the 5′ and 3′ donor insert/ELP genomic locus junctionswere completed. The results confirmed that the events identified via thedisruption assay and In-Out PCR analysis contained a donor insert withinthe ELP genomic locus.

Example 5 Analysis of Disrupted Loci in Maize Plants

Delivery of ZFNs to a genomic locus of a target line plant wereintroduced via a plant crossing strategy as previously disclosed in USPat. Pub. No. 20110191877. Separate target and excisor plant events weregenerated in Zea mays c.v. B104. Five lines of target plants wereproduced from transformations with constructs pDAB 105816, pDAB 105817,pDAB 105818, pDAB 105820, and pDAB 105821. These constructs contained astack of transgenes, an aad-1 selectable marker gene, and an ELPcontaining the eZF1 and eZF8 binding sites. Next, two lines of excisorplants were produced from transformation with constructs pDAB 105828 andpDAB 105825. The pDAB105828 construct contained ZFN8 driven by the Zeamays Ubiquitin 1 promoter and terminated by Zea mays Per5 3′UTR. ThepDAB105825 construct contained ZFN1 driven by the Zea mays Ubiquitin 1promoter and terminated by Zea mays Per5 3′UTR. Both of the excisorconstructs also contained a pat selectable marker. Transgenic plantswere produced and confirmed via molecular confirmation assays. Theplants were selfed to produce homozygous progeny. The resultinghomozygous target and excisor events were crossed producing progeny. Theprogeny were assayed to determine if the ZFN transgene provided by theparent excisor event cleaved the ELP target locus provided by the otherparent target event. The majority of all crosses were made with targetlines as females and excisor lines as males. Only one control cross wasmade with the excisor line as a female and the target line as a male.The target and excisor plants were crossed to produce progeny that werescreened by applying Assure II™ (quizalofop) (184 g ae/ha+1% COC) andIgnite™ 280 SL (glufosinate) (480 gae/ha) at the V3 stage ofdevelopment. Any surviving progeny were selected for further molecularanalysis that entailed screening for the presence of an aad-1 transgenevia a qPCR method. A total of 1902 samples were genotyped for thepresence of aad-1 locus using a qPCR assay utilizing invertase as thereference gene. The aad-1 target to reference ratios were calculated andnormalized to known standards, where a ratio of 2, 1, or 0 indicatedhomozygosity, hemizygosity, or null status respectively. 1822 plantswere confirmed as aad-1 hemizygous.

ZFN disruption assay: A PCR based disruption assay was designed for anindirect measurement of ZFN cutting activity of the ELP target genomiclocus. The assays were designed such that the fluorescently labeledprobes sat on top of the spacer required for the integrity of the ZFNfunctions (FIG. 10). The eZFN1 disruption assay resulted in thedetection of a 69 bp fragment covering the entire eZFN1 binding sitesequence. The eZFN8 disruption assay resulted in the detection of a 109bp fragment flanking the eZFN8 binding site sequence. Both assaysutilize MGB probes synthesized by Life Technologies (Grand Island,N.Y.). When ZFNs cleaved the ELP genomic locus the cleavage was repairedvia NHEJ which resulted in the incorporation of InDels within thegenomic sequences, thereby modifying the genomic sequence which had beenused for design of PCR primers. As a result any PCR amplificationreactions designed to amplify and detect an amplicon over the ZFNbinding sequence within the ELP genomic locus would not produce afluorescent signal (as the genomic sequences would be deleted orrearranged, and the primers could not bind these genomic sequences).

Bi-plex assays were performed with real-time or qPCR using theLightCycler®480 system (Roche Applied Science, Indianapolis, Ind.). Acontrol reaction was completed with Invertase used as an endogenousreference gene. For amplification, LightCycler®480 Probes Master mix(Roche Applied Science, Indianapolis, Ind.) was prepared at 1× finalconcentration in a 10 μL volume multiplex reaction containing 0.4 μM ofeach primer and 0.2 μM of each probe (Table 6). A three-stepamplification reaction started with 10 seconds at 95° C. fordenaturation, 35 seconds at 60° C. for annealing and 1 second at 72° C.for fluorescence acquisition. Analysis for the disruption assay wasperformed using target to reference ratios and normalized to knownhomozygotes. The results of the PCR assay are provided as FIG. 11.

TABLE 6 Primers used for qPCR detection. SEQ ID  Name NO: SequenceZFN1_F SEQ ID TAGTGAGATGG For eZFN1 NO: 27 GCGGGAGTCT detection ZFN1_PSEQ ID CCTAGTGGATA NO: 28 AACTGC ZFN1_R SEQ ID CCCACAGTGAT NO: 29CCGCCTTT ZFN8_F SEQ ID GCTTCTCTGTGA For eZFN8 NO: 30 TGATAACCCCTAdetection ZFN8_R SEQ ID TCCGCCTTTTGC NO: 31 AGTTTATC ZFN8S_P SEQ IDTGTCCCTAGTGA NO: 32 GATG InvertaseF SEQ ID TGGCGGACGACG Invertase NO: 33ACTTGT Reference  InvertaseProbe SEQ ID CGAGCAGACCG gene NO: 34CCGTGTACTT InvertaseR SEQ ID AAAGTTTGGAG NO: 35 GCTGCCGT aad-1F SEQ IDTGTTCGGTTC For aad-1 NO: 36 CCTCTACCAA control  aad-1P SEQ IDCACAGAACCGTC detection NO: 37 GCTTCAGCAACA aad-1R SEQ ID CAACATCCATCNO: 38 ACCTTGACTGA

To determine the eZFN cutting rate of the ELP genomic locus, F1 leafsamples from the plant crosses were first analyzed for zygosity and werethen tested for ELP disruption using a qPCR assay that detects intacteZFN binding sites. If the ELP genomic locus incurred InDels during DSBrepair on the eZF binding site, there was a loss or decrease indetectable signal in the qPCR assay. The normalized ELP ratios werecompared to their sister lines. If the ratio was between 0 to 0.05, theywere considered as being cut with the ZFN (with imperfect repair); ifthe ratio was between 0.05 to 0.4, they were labeled as chimeric,indicating not all copies of the ELPs have been cut with the ZFN; if theratio was over 0.4, they were identified as not being cut with the ZFN(could also have been cut but with perfect repair). Taking both cut andchimeric scoring together, eZFN1 activity detected with the disruptionassay was 46.6% (524 out of 1125 plant events) and the activity of eZFN8was 70.2% (489 out of 697 plant events). (Table 7). Tukey Kramer testrevealed significant differences among crosses for the cleavagefrequencies. The combination of L2BG and excisor lines proved crucialfor the success of cleavage (p<0.0001).

TABLE 7 ZFN cleavage activities among 5 ELP target lines and excisorline progeny. Crosses total cut chimeric no cut cut ratepDAB105821.1.295.1::pDAB105828.1.40.1 49 35 0 14 71.4%pDAB105821.1.295.1::pDAB105828.1.35.1 3 2 0 1 66.7%pDAB105821.1.295.1::pDAB105825.1.91.1 7 3 2 2 71.4%pDAB105821.1.295.1::pDAB105825.1.87.1 25 13 4 8 68.0%pDAB105821.1.295.1::pDAB105825.1.12.1 19 8 4 7 63.2%pDAB105821.1.264.1::pDAB105828.1.40.1 70 63 0 7 90.0%pDAB105821.1.264.1::pDAB105828.1.35.1 37 30 0 7 81.1%pDAB105821.1.264.1::pDAB105825.1.87.1 15 3 1 11 26.7%pDAB105821.1.264.1::pDAB105825.1.12.1 20 17 2 1 95.0%pDAB105820.1.199.1::pDAB105828.1.35.1 77 55 0 22 71.4%pDAB105820.1.199.1::pDAB105825.1.91.1 21 16 2 3 85.7%pDAB105820.1.199.1::pDAB105825.1.87.1 9 2 1 6 33.3%pDAB105828.1.40.1::pDAB105820.1.199.1 41 30 0 11 73.2%pDAB105820.1.140.1::pDAB105828.1.40.1 16 15 0 1 93.8%pDAB105820.1.140.1::pDAB105828.1.35.1 25 8 0 17 32.0%pDAB105820.1.140.1::pDAB105825.1.91.1 15 15 0 0 100.0%pDAB105820.1.140.1::pDAB105825.1.87.1 104 15 3 86 17.3%pDAB105820.1.140.1::pDAB105825.1.12.1 44 5 3 36 18.2%pDAB105818.2.388.1::pDAB105828.1.40.1 58 52 0 6 89.7%pDAB105818.2.388.1::pDAB105828.1.35.1 18 10 0 8 55.6%pDAB105818.2.388.1::pDAB105825.1.91.1 176 110 7 59 66.5%pDAB105818.2.388.1::pDAB105825.1.87.1 19 5 3 11 42.1%pDAB105818.2.388.1::pDAB105825.1.12.1 2 2 0 0 100.0%pDAB105818.1.269.1::pDAB105828.1.40.1 18 14 0 4 77.8%pDAB105818.1.269.1::pDAB105828.1.35.1 35 23 0 12 65.7%pDAB105818.1.269.1::pDAB105825.1.91.1 152 29 13 110 27.6%pDAB105818.1.269.1::pDAB105825.1.87.1 98 21 14 63 35.7%pDAB105818.1.269.1::pDAB105825.1.12.1 30 14 11 5 83.3%pDAB105817.1.81.1::pDAB105828.1.35.1 59 36 0 23 61.0%pDAB105817.1.81.1::pDAB105825.1.91.1 24 0 0 24 0.0%pDAB105817.1.81.1::pDAB105825.1.87.1 97 17 8 72 25.8%pDAB105817.1.81.1::pDAB105825.1.12.1 42 36 5 1 97.6%pDAB105817.1.6.1::pDAB105828.1.40.1 20 17 0 3 85.0%pDAB105817.1.6.1::pDAB105828.1.35.1 15 3 0 12 20.0%pDAB105817.1.6.1::pDAB105825.1.91.1 52 49 1 2 96.2%pDAB105816.2.496.1::pDAB105828.1.40.1 29 27 1 1 96.6%pDAB105816.2.496.1::pDAB105828.1.35.1 27 13 0 14 48.1%pDAB105816.2.496.1::pDAB105825.1.91.1 7 2 1 4 42.9%pDAB105816.2.496.1::pDAB105825.1.87.1 16 5 0 11 31.3%pDAB105816.2.496.1::pDAB105825.1.12.1 10 10 0 0 100.0%pDAB105816.2.447.1::pDAB105828.1.40.1 13 7 0 6 53.8%pDAB105816.2.447.1::pDAB105828.1.35.1 61 39 0 22 63.9%pDAB105816.2.447.1::pDAB105825.1.87.1 82 12 5 65 20.7%pDAB105821.1.309.1::pDAB105828.1.35.1 26 9 0 17 34.6%pDAB105821.1.309.1::pDAB105825.1.91.1 21 8 4 9 57.1%pDAB105821.1.309.1::pDAB105825.1.87.1 10 4 1 5 50.0%pDAB105821.1.309.1::pDAB105825.1.12.1 8 8 0 0 100.0%

Sequence analysis of ZFN cleavage: To confirm the qPCR based ELPdisruption data, representative samples from progeny crosses, and twonegative controls represented by the parental target lines were selectedfor NGS amplicon deep sequencing. High quality reads were alignedagainst the reference sequence from the ELP construct and insertionsand/or deletions (InDels) were identified. As expected, the two parentallines had very small percentage (<0.1%) of indels at the ZFN cleavagesite when comparing to the reference sequence, most likely due to PCRamplification and/or sequencing error (FIG. 12). There was a correlationbetween the qPCR and sequencing data. The four crosses exhibiting 100%cutting efficiencies based on the qPCR data had a 91% or more modifiedELPs with sequencing. In summary, NGS deep amplicon sequencing dataconcurred with the qPCR data and proved the disruption assay method tobe highly sensitive. In addition, the ELP disruption assay wasdemonstrated to be an effective asay for estimating the ZFN (or anyother site specific nuclease) cutting activities in planta.

While aspects of this invention have been described in certainembodiments, they can be further modified within the spirit and scope ofthis disclosure. This application is therefore intended to cover anyvariations, uses, or adaptations of embodiments of the invention usingits general principles. Further, this application is intended to coversuch departures from the present disclosure as come within known orcustomary practice in the art to which these embodiments pertain andwhich fall within the limits of the appended claims.

What is claimed is:
 1. A method for identifying the presence of anexogenous donor DNA polynucleotide inserted within a targeted genomiclocus comprising: a. amplifying in a first amplification reaction agenomic DNA sample comprising the targeted genomic locus using a firstplurality of oligonucleotides that bind under hybridization conditionsproximal to the targeted genomic locus, to thereby generate a firstamplicon comprising the targeted genomic locus; and, b. detecting thepresence or absence of the first amplicon, wherein the absence of thefirst amplicon indicates the presence of the donor DNA polynucleotidewithin the targeted genomic locus.
 2. The method of claim 1, the methodfurther comprising: a. amplifying in a second amplification reaction thegenomic DNA sample using a second plurality of oligonucleotides thatbind under hybridization conditions proximal to the targeted genomiclocus and within the donor DNA polynucleotide, to thereby generate asecond amplicon comprising at least a portion of the targeted genomiclocus and at least a portion of the donor DNA polynucleotide; and, b.detecting the presence or absence of the second amplicon, wherein thepresence of the second amplicon indicates the presence of the donor DNApolynucleotide within the targeted genomic locus.
 3. The method of claim2, the method further comprising: a. quantitating the results of thefirst amplification reaction; b. quantitating the results of the secondamplification reaction; c. comparing the results of the first and secondamplification reactions; and, d. determining the presence or absence ofthe donor DNA polynucleotide within the targeted genomic locus, whereinthe donor DNA polynucleotide is confirmed as inserted within thetargeted genomic locus if the first amplicon is absent and the secondamplicon is present.
 4. The method of claim 3, the method furthercomprising a multiplex reaction, wherein the first and secondamplification reactions are run in a single tube or well.
 5. The methodof claim 3, wherein quantitating the results of the of first and secondamplification reactions comprises producing a signature profile for oneor both of the first and second amplification reactions.
 6. Thesignature profile of claim 5, wherein the signature profile is selectedfrom the group consisting of a melting temperature curve signatureprofile and a fluorescence signature profile.
 7. The method of claim 5,wherein the signature profile is produced from an intercalating DNA dye.8. The method of claim 7, wherein the intercalating DNA dye comprises acyanine dye.
 9. The method of claim 8, wherein the cyanine dye is usedin an amplification reaction at a concentration of less than 4 μM. 10.The method of claim 8, wherein the cyanine dye is used in anamplification reaction at a concentration of less than 2.7 μM.
 11. Themethod of claim 5, wherein the signature profile is produced from afluorescent dye.
 12. The method of claim 1, the method furthercomprising: a. selecting a transgenic event comprising the donor DNApolynucleotide within the targeted genomic locus.
 13. A plant,comprising the transgenic event of claim
 12. 14. The plant of claim 13,wherein the plant is a dicot plant.
 15. The dicot plant of claim 14,wherein the dicot plant is selected from the group consisting of asoybean plant, a canola plant and a cotton plant.
 16. The plant of claim13, wherein the plant is a monocot plant.
 17. The monocot plant of claim17, wherein the monocot plant is selected from the group consisting of acorn plant, a rice plant, and a wheat plant.
 18. The method of claim 1,wherein the genomic locus is cleaved by a site specific nuclease. 19.The method of claim 18, wherein the nuclease comprises a zinc fingernuclease.
 20. The method of claim 18, wherein the nuclease comprises aTALEN or CRISPR nuclease.
 21. The method of claim 1, wherein theamplifying comprises amplifying in a polymerase chain reaction.
 22. Themethod of claim 2, wherein the second amplicon comprises a 5′ junctionof the donor DNA polynucleotide and the targeted genomic locus.
 23. Themethod of claim 2, wherein the second amplicon comprises a 3′ junctionof a donor DNA polynucleotide and the targeted genomic locus.
 24. Themethod of claim 1, wherein the donor DNA polynucleotide comprises atleast one gene expression cassette.
 25. The method of claim 1 or claim2, wherein the first or second plurality of oligonucleotides, or both,comprise a fluorescent dye.
 26. The method of claim 25, wherein thefluorescent dye is selected from the group consisting of a HEXfluorescent dye, a FAM fluorescent dye, a JOE fluorescent dye, a TETfluorescent dye, a Cy 3 fluorescent dye, a Cy 3.5 fluorescent dye, a Cy5 fluorescent dye, a Cy 5.5 fluorescent dye, a Cy 7 fluorescent dye, anda ROX fluorescent dye.
 27. A method for identifying a disruption of agenomic locus comprising: a. amplifying in a first amplificationreaction a genomic DNA sample comprising the disrupted genomic locususing a plurality of oligonucleotides that bind under hybridizationconditions proximal to the disrupted genomic locus, to thereby generatea first amplicon comprising the disrupted genomic locus; and, b.detecting the presence or absence of the first amplicon, wherein theabsence of the amplicon indicates the disruption of the genomic locus.28. The method of claim 27, the method further comprising: a.identifying the presence of a donor insertion within a disrupted genomiclocus; and, b. selecting a transgenic event comprising a donor insertionwithin a disrupted genomic locus.
 29. A plant, comprising the transgenicevent of claim
 28. 30. The plant of claim 29, wherein the plant is adicot plant.
 31. The dicot plant of claim 30, wherein the dicot plant isselected from the group consisting of a soybean plant, a canola plantand a cotton plant.
 32. The plant of claim 29, wherein the plant is amonocot plant.
 33. The monocot plant of claim 32, wherein the monocotplant is selected from the group consisting of a corn plant, a riceplant, and a wheat plant.
 34. The method of claim 27, wherein thegenomic locus is cleaved by a site specific nuclease.
 35. The method ofclaim 34, wherein the nuclease comprises a zinc finger nuclease.
 36. Themethod of claim 34, wherein the nuclease comprises a TALEN or CRISPRnuclease.
 37. The method of claim 27, wherein the amplifying comprisesamplifying in a polymerase chain reaction.
 38. The method of claim 27,wherein the donor DNA polynucleotide comprises at least one geneexpression cassette.
 39. The method of claim 27, wherein the pluralityof oligonucleotides, or both, comprise a fluorescent dye.
 40. The methodof claim 39, wherein the fluorescent dye is selected from the groupconsisting of a HEX fluorescent dye, a FAM fluorescent dye, a JOEfluorescent dye, a TET fluorescent dye, a Cy 3 fluorescent dye, a Cy 3.5fluorescent dye, a Cy 5 fluorescent dye, a Cy 5.5 fluorescent dye, a Cy7 fluorescent dye, and a ROX fluorescent dye.
 41. A method foridentifying a disruption of a genomic locus from a plurality of plantcells comprising: a. amplifying in a first amplification reaction agenomic DNA sample comprising the disrupted genomic locus using aplurality of oligonucleotides that bind under hybridization conditionsproximal to the disrupted genomic locus, to thereby generate a firstamplicon comprising the disrupted genomic locus; b. quantitating theresults of the first amplification reaction; c. amplifying in a secondamplification reaction a genomic DNA sample comprising the disruptedgenomic locus using the plurality of oligonucleotides that bind underhybridization conditions proximal to the disrupted genomic locus, tothereby generate a second amplicon comprising the disrupted genomiclocus; d. quantitating the results of the second amplification reaction;and, e. comparing the quantity of the first and second amplificationreactions, wherein the quantity of the first amplification reactioncomprises a lower quantity of amplified product as compared to thesecond amplification reaction thereby indicating the disruption of agenomic locus in the first amplicon samples.
 42. The method of claim 41,wherein quantitating the results of the of first and secondamplification reactions comprises producing a signature profile for oneor both of the first and second amplification reactions.
 43. Thesignature profile of claim 42, wherein the signature profile is selectedfrom the group consisting of a melting temperature curve signatureprofile and a fluorescence signature profile.
 44. The method of claim43, wherein the signature profile is produced from an intercalating DNAdye.
 45. The method of claim 44, wherein the intercalating DNA dyecomprises a cyanine dye.
 46. The method of claim 45, wherein the cyaninedye is used in an amplification reaction at a concentration of less than4 μM.
 47. The method of claim 45, wherein the cyanine dye is used in anamplification reaction at a concentration of less than 2.7 μM.
 48. Themethod of claim 42, wherein the signature profile is produced from afluorescent dye.
 49. The method of claim 41, the method furthercomprising: a. identifying the presence of a donor insertion within atargeted genomic locus; and, b. selecting a transgenic event comprisinga donor insertion within a targeted genomic locus.
 50. A plant,comprising the transgenic event of claim
 49. 51. The plant of claim 50,wherein the plant is a dicot plant.
 52. The dicot plant of claim 51,wherein the dicot plant is selected from the group consisting of asoybean plant, a canola plant and a cotton plant.
 53. The plant of claim50, wherein the plant is a monocot plant.
 54. The monocot plant of claim53, wherein the monocot plant is selected from the group consisting of acorn plant, a rice plant, and wheat plant.
 55. The method of claim 41,wherein the genomic locus is cleaved by a site specific nuclease. 56.The method of claim 55, wherein the nuclease comprises a zinc fingernuclease.
 57. The method of claim 55, wherein the nuclease comprises aTALEN or CRISPR nuclease.
 58. The method of claim 41, wherein theamplifying comprises amplifying in a polymerase chain reaction.
 59. Themethod of claim 41, wherein the donor DNA polynucleotide comprises atleast one gene expression cassette.
 60. The method of claim 41, whereinthe plurality of oligonucleotides comprise a fluorescent dye.
 61. Themethod of claim 60, wherein the fluorescent dye is selected from thegroup consisting of a HEX fluorescent dye, a FAM fluorescent dye, a JOEfluorescent dye, a TET fluorescent dye, a Cy 3 fluorescent dye, a Cy 3.5fluorescent dye, a Cy 5 fluorescent dye, a Cy 5.5 fluorescent dye, a Cy7 fluorescent dye, and a ROX fluorescent dye.